Multivalent clostridial toxins

ABSTRACT

The present invention is directed to multivalent Clostridial toxin comprising more than one binding domain directed to a cell surface molecule of a target cell. Such modified toxins are useful as therapeutic compositions to prevent exocytosis and secretion by the target cell. Conditions in which such compositions man be useful include, without limitation, disorders of the sensory or motor nervous system, acute or chronic pain, cancer, pancreatitis, hyperhydrosis, glandular disorders, viral infections, cystic fibrosis and the like. The invention is also directed to methods of using and administering such a composition, and methods of treating a given condition using such a composition.

This application is a continuation-in-part and claims priority pursuantto 35 U.S.C. § 120 to U.S. patent application Ser. No. 11/376,696, filedon Mar. 15, 2006, which is hereby incorporated by reference in itsentirety.

The ability of Clostridial toxins to inhibit neuronal transmission arebeing exploited in a wide variety of therapeutic and cosmeticapplications, see e.g., William J. Lipham, Cosmetic and ClinicalApplications of Botulinum Toxin (Slack, Inc., 2004). Clostridial toxinscommercially available as pharmaceutical compositions include, BoNT/Apreparations, such as, e.g., BOTOX® (Allergan, Inc., Irvine, Calif.),Dyspor®/Reloxin®, (Beaufour Ipsen, Porton Down, England), Linurase®(Prollenium, Inc., Ontario, Canada), Neuronox® (Medy-Tox, Inc.,Ochang-myeon, South Korea) BTX-A (Lanzhou Institute Biological Products,China) and Xeomin® (Merz Pharmaceuticals, GmbH, Frankfurt, Germany); andBoNT/B preparations, such as, e.g., MyoBloc™/NeuroBloc™ (ElanPharmaceuticals, San Francisco, Calif.). As an example, BOTOX® iscurrently approved in one or more countries for the followingindications: achalasia, adult spasticity, anal fissure, back pain,blepharospasm, bruxism, cervical dystonia, essential tremor, glabellarlines or hyperkinetic facial lines, headache, hemifacial spasm,hyperactivity of bladder, hyperhidrosis, juvenile cerebral palsy,multiple sclerosis, myoclonic disorders, nasal labial lines, spasmodicdysphonia, strabismus and VII nerve disorder.

Clostridial toxin therapies are successfully used for many indications.Generally, administration of a Clostridial toxin treatment is welltolerated. However, toxin administration in some applications can bechallenging because of the larger doses required to achieve a beneficialeffect. First, larger doses can increase the likelihood that the toxinmay move through the interstitial fluids and the circulatory systems,such as, e.g., the cardiovascular system and the lymphatic system, ofthe body, resulting in the undesirable dispersal of the toxin to areasnot targeted for toxin treatment. Such dispersal can lead to undesirableside effects, such as, e.g., inhibition of neurotransmitter release inneurons not targeted for treatment or paralysis of a muscle not targetedfor treatment. For example, a patient administered a therapeuticallyeffective amount of a BoNT/A treatment into the neck muscles fortorticollis may develop dysphagia because of dispersal of the toxin intothe oropharynx. Thus, there remains a need for improved Clostridialtoxins that are effective at the site of treatment, but have negligibleto minimal effects in areas not targeted for a toxin treatment.

Second, larger doses of a Clostridial toxin treatment may elicit anantibody response against the toxin. While a potent and effectivetreatment, the inhibition of neurotransmitter release and the resultingneuromuscular paralysis elicited by Clostridial toxin therapies is notpermanent. The reversible nature of these paralytic effects requiresperiodic treatments in order to maintain the therapeutic benefits fromthis toxin. As a consequence of this repeated exposure, an immuneresponse against a Clostridial toxin can occur in some patients whichreduce or completely prevent the individual's responsiveness to furthertreatments, see, e.g., Joseph Jankovic, Botulinum toxin: ClinicalImplications of Antigenicity and Immunoresistance, (SCIENTIFIC ANDTHERAPEUTIC ASPECTS OF BOTULINUM TOXIN, 409-415, Mitchell F. Brin etal., eds., Lippincott Williams & Wilkins, 2002); Dirk Dressler, ClinicalPresentation and Management of Antibody-induced Failure of BotulinumToxin Therapy, 19(Suppl. 8) MOV. DISORD. S92-S100 (2004); M. ZouhairAtassi, Basic Immunological Aspects of Botulinum Toxin Therapy,19(Suppl. 8) MOV. DISORD. S68-S84, (2004). Thus, there remains a needfor improved Clostridial toxins that maintain effective therapeuticbenefits, but have reduced ability to evoke an immunogenic responseagainst itself.

Moreover, a Clostridial toxin treatment inhibits neurotransmitterrelease by disrupting the exocytotic process used to secret theneurotransmitter into the synaptic cleft. However, it is believed thatcurrent Clostridial toxin therapies may by expanded to treat newindications beyond those diseases or disorders whose underlyingpathophysiology is aberrant cholinergic motor neuron activity.

Thus, the growing clinical, therapeutic and cosmetic use of Clostridialtoxins in therapies requiring larger doses necessitates thepharmaceutical industry to develop modified Clostridial toxins that areeffective at the target site of the application, but reduce or preventthe undesirable side-effects associated with the dispersal of the toxinsto unwanted locations and reduce or prevent an unwanted immunogenicresponse. Additionally, there is a great desire by the pharmaceuticalindustry to expand the use of Clostridial toxin therapies beyond itscurrent myo-relaxant applications to treat sensory-based ailment, suchas, e.g., various kinds of chronic pain, as well as non-neuronal baseddisorders, such as, e.g., pancreatitis. The present invention providesnovel multivalent Clostridial toxins that greatly extended the number oftherapeutic applications that can exploit the advantages offered bycurrent Clostridial toxin therapies. These and related advantages areuseful for various clinical, therapeutic and cosmetic applications, suchas, e.g., the treatment of neuropathic disorders, eye disorders, pain,muscle injuries, headache, cardiovascular diseases, neuropsychiatricdisorders, endocrine disorders, cancers, otic disorders, as well as,other disorders where administration of a multivalent Clostridial toxinto an individual can produce a beneficial effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the current paradigm of neurotransmitterrelease and Clostridial toxin intoxication in a central and peripheralneuron. FIG. 1A shows a schematic for the neurotransmitter releasemechanism of a central and peripheral neuron. The release process can bedescribed as comprising two steps: 1) vesicle docking, where thevesicle-bound SNARE protein of a vesicle containing neurotransmittermolecules associates with the membrane-bound SNARE proteins located atthe plasma membrane; and 2) neurotransmitter release, where the vesiclefuses with the plasma membrane and the neurotransmitter molecules areexocytosed. FIG. 1B shows a schematic of the intoxication mechanism fortetanus and botulinum toxin activity in a central and peripheral neuron.This intoxication process can be described as comprising four steps: 1)receptor binding, where a Clostridial toxin binds to a Clostridialreceptor and initiates the intoxication process; 2) complexinternalization, where after toxin binding, a vesicle containing thetoxin/receptor complex is endocytosed into the cell; 3) light chaintranslocation, where multiple events result in the release of the activelight chain into the cytoplasm; and 4) enzymatic target modification,where the active light chain of Clostridial toxin proteolyticallycleaves its target SNARE substrate, such as, e.g., SNAP-25, VAMP orSyntaxin, thereby preventing vesicle docking and neurotransmitterrelease.

FIG. 2 shows the domain organization of naturally-occurring Clostridialtoxins. The single chain form depicts the amino to carboxyl linearorganization comprising an enzymatic domain, a translocation domain, aH_(CN) translocation facilitating domain and a H_(CC) targeting domain.The di-chain loop region located between the translocation and enzymaticdomains is depicted by the double SS bracket. This region comprises anendogenous di-chain loop protease cleavage site that upon proteolyticcleavage with a naturally-occurring protease, such as, e.g., anendogenous Clostridial toxin protease or a naturally-occurring proteaseproduced in the environment, converts the single chain form of the toxininto the di-chain form. As depicted above the single-chain form, theH_(CC) targeting domain comprises the β-trefoil domain which comprisesin an amino to carboxyl linear organization of an α-fold, a β4/β5hairpin turn, a β-fold, a β8/β9 hairpin turn and a γ-fold.

FIG. 3 shows a ribbon diagram of BoNT/A illustrating the modularthree-dimensional structure of the light chain (LC) comprising theenzymatic domain, the heavy chain H_(N) domain comprising thetranslocation domain, and the heavy chain H_(CN) domain, including theheavy chain H_(CN) domain and the heavy chain H_(CC) domain, thatcomprises the binding domain.

FIG. 4 shows examples of domain arrangements of multivalent Clostridialtoxins. FIG. 4A depicts the single polypeptide form of a multivalentClostridial toxin with an amino to carboxyl linear organizationcomprising a binding domain 1, a translocation domain, a binding domain2 and an enzymatic domain, with the di-chain loop region depicted by thedouble SS bracket. A proteolytic cleavage site (P) within a di-chainloop region is located between the binding domain 2 and enzymaticdomain. Upon proteolytic cleavage with a P protease, the single chainform of the toxin is converted to the di-chain form. The P protease sitecan be a Clostridial toxin endogenous protease cleavage site or anon-Clostridial toxin exogenous protease cleavage site. Spacers can beplaced between the binding domain 1 and translocation domain, thetranslocation domain and binding domain 2, binding domain 2 andenzymatic domain, or any combination thereof. FIG. 4B depicts the singlepolypeptide form of a multivalent Clostridial toxin with an amino tocarboxyl linear organization comprising an enzymatic domain, a bindingdomain 1, a translocation domain and a binding domain 2, with thedi-chain loop region depicted by the double SS bracket. A proteolyticcleavage site (P) within a di-chain loop region is located between theenzymatic domain and binding domain 1. Upon proteolytic cleavage with aP protease, the single chain form of the toxin is converted to thedi-chain form. The P protease site can be a Clostridial toxin endogenousprotease cleavage site or a non-Clostridial toxin exogenous proteasecleavage site. Spacers can be placed between the enzymatic domain andbinding domain 1, the binding domain 1 and translocation domain, thetranslocation and binding domain 2, or any combination thereof. FIG. 4Cdepicts the single polypeptide form of a multivalent Clostridial toxinwith an amino to carboxyl linear organization comprising an enzymaticdomain, a translocation domain, a binding domain 1 and a binding domain2, with the di-chain loop region depicted by the double SS bracket. Aproteolytic cleavage site (P) within a di-chain loop region is locatedbetween the enzymatic domain and translocation domain. Upon proteolyticcleavage with a P protease, the single chain form of the toxin isconverted to the di-chain form. The P protease site can be a Clostridialtoxin endogenous protease cleavage site or a non-Clostridial toxinexogenous protease cleavage site. Spacers can be placed between theenzymatic domain and translocation domain, the translocation domain andbinding domain 1, the binding domain 1 and a binding domain 2, or anycombination thereof. FIG. 4D depicts the single polypeptide form of amultivalent Clostridial toxin with an amino to carboxyl linearorganization comprising an enzymatic domain, a translocation domain, abinding domain 2 and a binding domain 1, with the di-chain loop regiondepicted by the double SS bracket. A proteolytic cleavage site (P)within a di-chain loop region is located between the enzymatic domainand translocation domain. Upon proteolytic cleavage with a P protease,the single chain form of the toxin is converted to the di-chain form.The P protease site can be a Clostridial toxin endogenous proteasecleavage site or a non-Clostridial toxin exogenous protease cleavagesite. Spacers can be placed between the enzymatic domain andtranslocation domain, the translocation domain and binding domain 2, thebinding domain 2 and a binding domain 1, or any combination thereof.

FIG. 5 shows examples of domain arrangements of multivalent Clostridialtoxins. FIG. 5A depicts the single polypeptide form of a multivalentClostridial toxin with an amino to carboxyl linear organizationcomprising a binding domain 1, an enzymatic domain, a translocationdomain and a binding domain 2, with the di-chain loop region depicted bythe double SS bracket. A proteolytic cleavage site (P) within a di-chainloop region is located between the enzymatic domain and translocationdomain. Upon proteolytic cleavage with a P protease, the single chainform of the toxin is converted to the di-chain form. The P protease sitecan be a Clostridial toxin endogenous protease cleavage site or anon-Clostridial toxin exogenous protease cleavage site. Spacers can beplaced between the binding domain 1 and the enzymatic domain, theenzymatic domain and translocation domain, the translocation domain andbinding domain 2, or any combination thereof. FIG. 5B depicts the singlepolypeptide form of a multivalent Clostridial toxin with an amino tocarboxyl linear organization comprising a translocation domain, abinding domain 2, a binding domain 1 and an enzymatic domain, with thedi-chain loop region depicted by the double SS bracket. A proteolyticcleavage site (P) within a di-chain loop region is located between thebinding domain 2 and binding domain 1. Upon proteolytic cleavage with aP protease, the single chain form of the toxin is converted to thedi-chain form. The P protease site can be a Clostridial toxin endogenousprotease cleavage site or a non-Clostridial toxin exogenous proteasecleavage site. Spacers can be placed between the translocation domainand binding domain 2, the binding domain 2 and binding domain 1, thebinding domain 1 and enzymatic domain, or any combination thereof. FIG.5C depicts the single polypeptide form of a multivalent Clostridialtoxin with an amino to carboxyl linear organization comprising atranslocation domain, a binding domain 2, an enzymatic domain and abinding domain 1, with the di-chain loop region depicted by the doubleSS bracket. A proteolytic cleavage site (P) within a di-chain loopregion is located between the binding domain 2 and enzymatic domain.Upon proteolytic cleavage with a P protease, the single chain form ofthe toxin is converted to the di-chain form. The P protease site can bea Clostridial toxin endogenous protease cleavage site or anon-Clostridial toxin exogenous protease cleavage site. Spacers can beplaced between the translocation domain and binding domain 2, thebinding domain 2 and enzymatic domain, the enzymatic domain and bindingdomain 1, or any combination thereof. FIG. 5D depicts the singlepolypeptide form of a multivalent Clostridial toxin with an amino tocarboxyl linear organization comprising a binding domain 2, atranslocation domain, a binding domain 1 and an enzymatic domain, withthe di-chain loop region depicted by the double SS bracket. Aproteolytic cleavage site (P) within a di-chain loop region is locatedbetween the translocation domain and binding domain 1. Upon proteolyticcleavage with a P protease, the single chain form of the toxin isconverted to the di-chain form. The P protease site can be a Clostridialtoxin endogenous protease cleavage site or a non-Clostridial toxinexogenous protease cleavage site. Spacers can be placed between thebinding domain 2 and translocation domain, the translocation domain andbinding domain 1, the binding domain 1 and enzymatic domain, or anycombination thereof.

FIG. 6 shows examples of domain arrangements of multivalent Clostridialtoxins. FIG. 6A depicts the single polypeptide form of a multivalentClostridial toxin with an amino to carboxyl linear organizationcomprising an enzymatic domain, a translocation domain, a binding domain1 and a binding domain 2, with the di-chain loop region depicted by thedouble SS bracket. A first protease cleavage site (P) within a di-chainloop region is located between the enzymatic domain and translocationdomain. A second protease cleavage site (P) is located between thebinding domain 1 and binding domain 2. Upon proteolytic cleavage with aP protease, the single chain form of the toxin is converted to thetri-chain form. The P protease site can be a Clostridial toxinendogenous protease cleavage site or a non-Clostridial toxin exogenousprotease cleavage site. Additionally, the first and second proteolyticcleavage sites can be the same site cleaved by the same protease ordifferent sites cleaved by different proteases. Spacers can be placedbetween the enzymatic domain and translocation domain, the translocationdomain and binding domain 1, the binding domain 1 and binding domain 2,or any combination thereof. FIG. 6B depicts the single polypeptide formof a multivalent Clostridial toxin with an amino to carboxyl linearorganization comprising an enzymatic domain, a translocation domain, abinding domain 2 and a binding domain 1, with the di-chain loop regiondepicted by the double SS bracket. A first protease cleavage site (P)within a di-chain loop region is located between the enzymatic domainand translocation domain. A second protease cleavage site (P) is locatedbetween the binding domain 2 and binding domain 1. Upon proteolyticcleavage with a P protease, the single chain form of the toxin isconverted to the tri-chain form. The P protease cleavage site can be aClostridial toxin endogenous protease cleavage site or a non-Clostridialtoxin exogenous protease cleavage site. Additionally, the first andsecond protease cleavage sites can be the same site cleaved by the sameprotease or different sites cleaved by different proteases. Spacers canbe placed between the enzymatic domain and translocation domain, thetranslocation domain and binding domain 2, the binding domain 2 andbinding domain 1, or any combination thereof.

DETAILED DESCRIPTION

The present invention provides novel Clostridial toxins that greatlyextended the number of therapeutic applications that can exploit theadvantages offered by current Clostridial toxin therapies. Thesemultivalent Clostridial toxins comprise, in part, multiple bindingdomains. Each binding domain can be capable of binding 1) the cognateClostridial toxin receptor present on the surface of anaturally-occurring Clostridial toxin target cell; 2) a differentreceptor present on the surface of a naturally occurring Clostridialtoxin target cell; or 3) a different receptor present on the surface ofthe non-Clostridial toxin target cell. As such, a multivalentClostridial toxin comprising multiple binding domains can exhibitincreased specificity, efficacy and efficiency by which such multivalentClostridial toxins can interact with a particular target cell andenzymatically modify its target SNARE substrate. Additionally, amultivalent Clostridial toxin comprising multiple binding domains canexhibit increased the versatility and therapeutic scope of currentClostridial toxin therapeutic applications by simultaneously targetingmultiple cell types responsible for different aspects of a diseasedstate, such as, e.g., a nerve spasticity symptom and a pain symptom, oran aberrant enzyme release symptom and a pain symptom.

Aspects of the present invention a composition comprising a multivalentClostridial toxin comprising a Clostridial toxin enzymatic domain, aClostridial toxin translocation domain, a first binding domain and asecond binding domain, wherein each of the binding domains isindependently capable of binding a cell surface receptor of a targetcell. It is envisioned that the first binding domain and second bindingdomain may be identical to each other or different.

Other aspects of the present invention provide polynucleotide moleculesencoding a multivalent Clostridial toxin comprising a Clostridial toxinenzymatic domain, a Clostridial toxin translocation domain, a firstbinding domain and a second binding domain.

Other aspects of the present invention provide methods of producing amultivalent Clostridial toxin disclosed in the present specification,the method comprising the step of expressing in a cell a polynucleotidemolecule encoding a multivalent Clostridial toxin comprising aClostridial toxin enzymatic domain, a Clostridial toxin translocationdomain, a first binding domain and a second binding domain. Otheraspects of the present invention provide methods of producing amultivalent Clostridial toxin disclosed in the present specification,the method comprising the steps of introducing in a cell an expressionconstruct comprising a polynucleotide molecule encoding a multivalentClostridial toxin comprising a Clostridial toxin enzymatic domain, aClostridial toxin translocation domain, a first binding domain and asecond binding domain and expressing the expression construct in thecell.

Clostridia toxins produced by Clostridium botulinum, Clostridium tetani,Clostridium baratii and Clostridium butyricum are the most widely usedin therapeutic and cosmetic treatments of humans and other mammals.Strains of C. botulinum produce seven antigenically-distinct types ofBotulinum toxins (BoNTs), which have been identified by investigatingbotulism outbreaks in man (BoNT/A, /B, /E and /F), animals (BoNT/C1 and/D), or isolated from soil (BoNT/G). BoNTs possess approximately 35%amino acid identity with each other and share the same functional domainorganization and overall structural architecture. It is recognized bythose of skill in the art that within each type of Clostridial toxinthere can be subtypes that differ somewhat in their amino acid sequence,and also in the nucleic acids encoding these proteins. For example,there are presently four BoNT/A subtypes, BoNT/A1, BoNT/A2, BoNT/A3 andBoNT/A4, with specific subtypes showing approximately 89% amino acididentity when compared to another BoNT/A subtype. While all seven BoNTserotypes have similar structure and pharmacological properties, eachalso displays heterogeneous bacteriological characteristics. Incontrast, tetanus toxin (TeNT) is produced by a uniform group of C.tetani. Two other species of Clostridia, C. baratii and C. butyricum,also produce toxins, BaNT and BuNT respectively, which are similar toBoNT/F and BoNT/E, respectively.

Clostridial toxins are each translated as a single chain polypeptide ofapproximately 150 kDa that is subsequently cleaved by proteolyticscission within a disulfide loop by a naturally-occurring protease (FIG.2). This cleavage occurs within the discrete di-chain loop regioncreated between two cysteine residues that form a disulfide bridge. Thisposttranslational processing yields a di-chain molecule comprising anapproximately 50 kDa light chain (LC) and an approximately 100 kDa heavychain (HC) held together by the single disulfide bond and non-covalentinteractions between the two chains. The naturally-occurring proteaseused to convert the single chain molecule into the di-chain is currentlynot known. In some serotypes, such as, e.g., BoNT/A, thenaturally-occurring protease is produced endogenously by the bacteriaserotype and cleavage occurs within the cell before the toxin is releaseinto the environment. However, in other serotypes, such as, e.g.,BoNT/E, the bacterial strain appears not to produce an endogenousprotease capable of converting the single chain form of the toxin intothe di-chain form. In these situations, the toxin is released from thecell as a single-chain toxin which is subsequently converted into thedi-chain form by a naturally-occurring protease found in theenvironment.

TABLE 1 Clostridial Toxin Reference Sequences and Regions H_(C) ToxinSEQ ID NO: LC H_(N) H_(CN) H_(CC) BoNT/A 1 M1-K448 A449-I873 I874-P1110Y1111-L1296 BoNT/B 2 M1-K441 A442-I860 L861-E1097 Y1098-E1291 BoNT/C1 3M1-K449 T450-I868 N869-E1111 Y1112-E1291 BoNT/D 4 M1-R445 D446-I864N865-E1098 Y1099-E1276 BoNT/E 5 M1-R422 K423-I847 K848-E1085 Y1086-K1252BoNT/F 6 M1-K439 A440-I866 K867-K1105 Y1106-E1274 BoNT/G 7 M1-K446S447-I865 S866-Q1105 Y1106-E1297 TeNT 8 M1-A457 S458-L881 K882-E1127Y1128-D1315 BaNT 9 M1-K431 N432-I857 I858-K1094 Y1095-E1268 BuNT 10 M1-R422 K423-I847 K848-E1085 Y1086-K1251

Each mature di-chain molecule comprises three functionally distinctdomains: 1) an enzymatic domain located in the LC that includes ametalloprotease region containing a zinc-dependent endopeptidaseactivity which specifically targets core components of theneurotransmitter release apparatus; 2) a translocation domain containedwithin the amino-terminal half of the HC(H_(N)) that facilitates releaseof the LC from intracellular vesicles into the cytoplasm of the targetcell; and 3) a binding domain found within the carboxyl-terminal half ofthe HC (H_(C)) that determines the binding activity and bindingspecificity of the toxin to the receptor complex located at the surfaceof the target cell. The H_(C) domain comprises two distinct structuralfeatures of roughly equal size that indicate function and are designatedthe H_(CN) and H_(CC) subdomains. Table 1 gives approximate boundaryregions for each domain and subdomain found in exemplary Clostridialtoxins. When discussing the three general neurotoxin domains of eachclostridial neurotoxin subtype (binding, translocation andendopeptidase) it will be understood that clostridial neurotoxinresearch is a well-developed field, and the correlation of the aminoacid sequences comprising each of these domains with their functions iswell known. Additionally, the nucleotide and amino acid sequences ofeach of these domains are known and have been disclosed in thisspecification.

The binding, translocation and enzymatic activity of these threefunctional domains are all necessary for toxicity. While all details ofthis process are not yet precisely known, the overall cellularintoxication mechanism whereby Clostridial toxins enter a neuron andinhibit neurotransmitter release is similar, regardless of type.Although the applicants have no wish to be limited by the followingdescription, the intoxication mechanism can be described as comprisingat least four steps: 1) receptor binding, 2) complex internalization, 3)light chain translocation, and 4) enzymatic target modification (seeFIG. 1). The process is initiated when the H_(C) domain of a Clostridialtoxin binds to a toxin-specific receptor complex located on the plasmamembrane surface of a target cell. The binding specificity of a receptorcomplex is thought to be achieved, in part, by specific combinations ofgangliosides and protein receptors that appear to distinctly compriseeach Clostridial toxin receptor complex. Once bound, the toxin/receptorcomplexes are internalized by endocytosis and the internalized vesiclesare sorted to specific intracellular routes. The translocation stepappears to be triggered by the acidification of the vesicle compartment.This process seems to initiate two important pH-dependent structuralrearrangements that increase hydrophobicity and promote formationdi-chain form of the toxin. Once activated, light chain endopeptidase ofthe toxin is released from the intracellular vesicle into the cytosolwhere it specifically targets one of three known core components of theneurotransmitter release apparatus. These core proteins,vesicle-associated membrane protein (VAMP)/synaptobrevin,synaptosomal-associated protein of 25 kDa (SNAP-25) and Syntaxin, arenecessary for synaptic vesicle docking and fusion at the nerve terminaland constitute members of the soluble N-ethylmaleimide-sensitivefactor-attachment protein-receptor (SNARE) family. BoNT/A and BoNT/Ecleave SNAP-25 in the carboxyl-terminal region, releasing a nine ortwenty-six amino acid segment, respectively, and BoNT/C1 also cleavesSNAP-25 near the carboxyl-terminus. The botulinum serotypes BoNT/B,BoNT/D, BoNT/F and BoNT/G, and tetanus toxin, act on the conservedcentral portion of VAMP, and release the amino-terminal portion of VAMPinto the cytosol. BoNT/C1 cleaves syntaxin at a single site near thecytosolic membrane surface. The selective proteolysis of synaptic SNAREsaccounts for the block of neurotransmitter release caused by Clostridialtoxins in vivo. The SNARE protein targets of Clostridial toxins arecommon to exocytosis in a variety of non-neuronal types; in these cells,as in neurons, light chain peptidase activity inhibits exocytosis, see,e.g., Yann Humeau et al., How Botulinum and Tetanus Neurotoxins BlockNeurotransmitter Release, 82(5) Biochimie. 427-446 (2000); KathrynTurton et al., Botulinum and Tetanus Neurotoxins: Structure, Functionand Therapeutic Utility, 27(11) Trends Biochem. Sci. 552-558. (2002);Giovanna Lalli et al., The Journey of Tetanus and Botulinum Neurotoxinsin Neurons, 11(9) Trends Microbiol. 431-437, (2003).

The three-dimensional crystal structures of BoNT/A, BoNT/B and the H_(C)domain of TeNT indicate that the three functional domains of Clostridialneurotoxins are structurally distinct domains that are shared by allClostridial toxins. The HEXXH consensus motif of the light chain formsthe tetrahedral zinc binding pocket of the catalytic site located in adeep cleft on the protein surface that is accessible by a channel. Thestructure of the H_(N) and H_(C) domains consists primarily of β-sheettopologies that are linked by a single α-helix. The cylindrical-shapedH_(N) domain comprises two long amphipathic α-helices that resemble thecoiled-coil motif found in some viral proteins. The H_(N) domain alsoforms a long unstructured loop called the ‘translocation belt,’ whichwraps around a large negatively charged cleft of the light chain thatblocks access of the zinc atom to the catalytic-binding pocket of activesite. The H_(C) domain comprises two distinct structural features ofroughly equal size that indicate function. The first, designated theH_(CN) domain, is located in the amino half of the H_(C) domain. TheH_(CN) domain forms a β-barrel, jelly-roll fold. The H_(CC) domain isthe second domain that comprises the H_(C) domain. Thiscarboxyl-terminal domain comprises a modified β-trefoil domain whichforms three distinct carbohydrate binding regions that resembles thecarbohydrate binding moiety found in many sugar-binding proteins, suchas, e.g., serum amyloid P, sialidase, cryia, insecticidal ∂-endotoxinand lectins. Biochemical studies indicate that the β-trefoil domainstructure of the H_(CC) domain appears to mediate the binding tospecific carbohydrate containing components of the Clostridial toxinreceptor on the cell surface, see, e.g., Krzysztof Ginalski et al.,Structure-based Sequence Alignment for the Beta-Trefoil Subdomain of theClostridial Neurotoxin Family Provides Residue Level Information Aboutthe Putative Ganglioside Binding Site, 482(1-2) FEBS Lett. 119-124(2000). The H_(C) domain tilts away from the H_(N) domain exposing thesurface loops and making them accessible for binding. No contacts occurbetween the light chain and the H_(C) domain.

Aspects of the present invention provide, in part, a Clostridial toxin.As used herein, the term “Clostridial toxin” means any neurotoxinproduced by a Clostridial toxin strain that can execute the overallcellular mechanism whereby a Clostridial toxin intoxicates a cell andencompasses the binding of a Clostridial toxin to a low or high affinityreceptor complex, the internalization of the toxin/receptor complex, thetranslocation of the Clostridial toxin light chain into the cytoplasmand the enzymatic modification of a Clostridial toxin substrate.Exemplary Clostridial toxins include those produced by a Clostridiumbotulinum, a Clostridium tetani, a Clostridium baratii and a Clostridiumbutyricum.

A Clostridial toxin includes, without limitation, naturally occurringClostridial toxin variants, such as, e.g., Clostridial toxin isoformsand Clostridial toxin subtypes; non-naturally occurring Clostridialtoxin variants, such as, e.g., conservative Clostridial toxin variants,non-conservative Clostridial toxin variants, Clostridial toxin chimericvariants and active Clostridial toxin fragments thereof, or anycombination thereof. As used herein, the term “Clostridial toxinvariant,” whether naturally-occurring or non-naturally-occurring, meansa Clostridial toxin that has at least one amino acid change from thecorresponding region of the disclosed reference sequences (see Table 1)and can be described in percent identity to the corresponding region ofthat reference sequence. As non-limiting examples, a BoNT/A variantcomprising amino acids 1-1296 of SEQ ID NO: 1 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 1-1296 of SEQID NO: 1; a BoNT/B variant comprising amino acids 1-1291 of SEQ ID NO: 2will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 1-1291 of SEQ ID NO: 2; a BoNT/C1 variant comprising amino acids1-1291 of SEQ ID NO: 3 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 1-1291 of SEQ ID NO: 3; a BoNT/Dvariant comprising amino acids 1-1276 of SEQ ID NO: 4 will have at leastone amino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 1-1276 of SEQID NO: 4; a BoNT/E variant comprising amino acids 1-1252 of SEQ ID NO: 5will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 1-1252 of SEQ ID NO: 5; a BoNT/F variant comprising amino acids1-1274 of SEQ ID NO: 6 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 1-1274 of SEQ ID NO: 6; a BoNT/Gvariant comprising amino acids 1-1297 of SEQ ID NO: 7 will have at leastone amino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 1-1297 of SEQID NO: 7; a TeNT variant comprising amino acids 1-1315 of SEQ ID NO: 8will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 1-1315 of SEQ ID NO: 8; a BaNT variant comprising amino acids1-1268 of SEQ ID NO: 9 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 1-1268 of SEQ ID NO: 9; and a BuNTvariant comprising amino acids 1-1251 of SEQ ID NO: 10 will have atleast one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region1-1251 of SEQ ID NO: 10.

Any of a variety of sequence alignment methods can be used to determinepercent identity, including, without limitation, global methods, localmethods and hybrid methods, such as, e.g., segment approach methods.Protocols to determine percent identity are routine procedures withinthe scope of one skilled in the art and from the teaching herein.

Global methods align sequences from the beginning to the end of themolecule and determine the best alignment by adding up scores ofindividual residue pairs and by imposing gap penalties. Non-limitingmethods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al.,CLUSTAL W: Improving the Sensitivity of Progressive Multiple SequenceAlignment Through Sequence Weighting, Position-Specific Gap Penaltiesand Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680(1994); and iterative refinement, see, e.g., Osamu Gotoh, SignificantImprovement in Accuracy of Multiple Protein Sequence Alignments byIterative Refinement as Assessed by Reference to Structural Alignments,264(4) J. Mol. Biol. 823-838 (1996).

Local methods align sequences by identifying one or more conservedmotifs shared by all of the input sequences. Non-limiting methodsinclude, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans,Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignmentof Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbssampling, see, e.g., C. E. Lawrence et al., Detecting Subtle SequenceSignals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131)Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al.,Align-M—A New Algorithm for Multiple Alignment of Highly DivergentSequences, 20(9) Bioinformatics:1428-1435 (2004).

Hybrid methods combine functional aspects of both global and localalignment methods. Non-limiting methods include, e.g.,segment-to-segment comparison, see, e.g., Burkhard Morgenstern et al.,Multiple DNA and Protein Sequence Alignment Based On Segment-To-SegmentComparison, 93(22) Proc. Natl. Acad. Sci. U.S.A. 12098-12103 (1996);T-Coffee, see, e.g., Cédric Notredame et al., T-Coffee: A NovelAlgorithm for Multiple Sequence Alignment, 302(1) J. Mol. Biol. 205-217(2000); MUSCLE, see, e.g., Robert C. Edgar, MUSCLE: Multiple SequenceAlignment With High Score Accuracy and High Throughput, 32(5) NucleicAcids Res. 1792-1797 (2004); and DIALIGN-T, see, e.g., Amarendran RSubramanian et al., DIALIGN-T: An Improved Algorithm for Segment-BasedMultiple Sequence Alignment, 6(1) BMC Bioinformatics 66 (2005).

As used herein, the term “naturally occurring Clostridial toxin variant”means any Clostridial toxin produced without the aid of any humanmanipulation, including, without limitation, Clostridial toxin isoformsproduced from alternatively-spliced transcripts, Clostridial toxinisoforms produced by spontaneous mutation and Clostridial toxinsubtypes. Non-limiting examples of a Clostridial toxin isoform include,e.g., BoNT/A isoforms, BoNT/β isoforms, BoNT/C1 isoforms, BoNT/Disoforms, BoNT/E isoforms, BoNT/F isoforms, BoNT/G isoforms, TeNTisoforms, BaNT isoforms and BuNT isoforms. Non-limiting examples of aClostridial toxin subtype include, e.g., BoNT/A subtypes BoNT/A1,BoNT/A2, BoNT/A3 and BoNT/A4; BoNT/B subtypes BoNT/B1, BoNT/B2, BoNT/Bbivalent and BoNT/B nonproteolytic; BoNT/C1 subtypes BoNT/C1-1 andBoNT/C1-2; BoNT/E subtypes BoNT/E1, BoNT/E2 and BoNT/E3; and BoNT/Fsubtypes BoNT/F1, BoNT/F2, BoNT/F3 and BoNT/F4.

As used herein, the term “non-naturally occurring Clostridial toxinvariant” means any Clostridial toxin produced with the aid of humanmanipulation, including, without limitation, Clostridial toxins producedby genetic engineering using random mutagenesis or rational design andClostridial toxins produced by chemical synthesis. Non-limiting examplesof non-naturally occurring Clostridial toxin variants include, e.g.,conservative Clostridial toxin variants, non-conservative Clostridialtoxin variants, Clostridial toxin chimeric variants and activeClostridial toxin fragments.

As used herein, the term “conservative Clostridial toxin variant” meansa Clostridial toxin that has at least one amino acid substituted byanother amino acid or an amino acid analog that has at least oneproperty similar to that of the original amino acid from the referenceClostridial toxin sequence (Table 1). Examples of properties include,without limitation, similar size, topography, charge, hydrophobicity,hydrophilicity, lipophilicity, covalent-bonding capacity,hydrogen-bonding capacity, a physicochemical property, of the like, orany combination thereof. A conservative Clostridial toxin variant canfunction in substantially the same manner as the reference Clostridialtoxin on which the conservative Clostridial toxin variant is based, andcan be substituted for the reference Clostridial toxin in any aspect ofthe present invention. A conservative Clostridial toxin variant maysubstitute one or more amino acids, two or more amino acids, three ormore amino acids, four or more amino acids, five or more amino acids,ten or more amino acids, 20 or more amino acids, 30 or more amino acids,40 or more amino acids, 50 or more amino acids, 100 or more amino acids,200 or more amino acids, 300 or more amino acids, 400 or more aminoacids, or 500 or more amino acids from the reference Clostridial toxinon which the conservative Clostridial toxin variant is based. Aconservative Clostridial toxin variant can also substitute at least 10contiguous amino acids, at least 15 contiguous amino acids, at least 20contiguous amino acids, or at least 25 contiguous amino acids from thereference Clostridial toxin on which the conservative Clostridial toxinvariant is based, that possess at least 50% amino acid identity, 65%amino acid identity, 75% amino acid identity, 85% amino acid identity or95% amino acid identity to the reference Clostridial toxin on which theconservative Clostridial toxin variant is based. Non-limiting examplesof a conservative Clostridial toxin variant include, e.g., conservativeBoNT/A variants, conservative BoNT/B variants, conservative BoNT/C1variants, conservative BoNT/D variants, conservative BoNT/E variants,conservative BoNT/F variants, conservative BoNT/G variants, conservativeTeNT variants, conservative BaNT variants and conservative BuNTvariants.

As used herein, the term “non-conservative Clostridial toxin variant”means a Clostridial toxin in which 1) at least one amino acid is deletedfrom the reference Clostridial toxin on which the non-conservativeClostridial toxin variant is based; 2) at least one amino acid added tothe reference Clostridial toxin on which the non-conservativeClostridial toxin is based; or 3) at least one amino acid is substitutedby another amino acid or an amino acid analog that does not share anyproperty similar to t h at of the original amino acid from the referenceClostridial toxin sequence (Table 1). A non-conservative Clostridialtoxin variant can function in substantially the same manner as thereference Clostridial toxin on which the non-conservative Clostridialtoxin variant is based, and can be substituted for the referenceClostridial toxin in any aspect of the present invention. Anon-conservative Clostridial toxin variant can delete one or more aminoacids, two or more amino acids, three or more amino acids, four or moreamino acids, five or more amino acids, and ten or more amino acids fromthe reference Clostridial toxin on which the non-conservativeClostridial toxin variant is based. A non-conservative Clostridial toxinvariant can add one or more amino acids, two or more amino acids, threeor more amino acids, four or more amino acids, five or more amino acids,and ten or more amino acids to the reference Clostridial toxin on whichthe non-conservative Clostridial toxin variant is based. Anon-conservative Clostridial toxin variant may substitute one or moreamino acids, two or more amino acids, three or more amino acids, four ormore amino acids, five or more amino acids, ten or more amino acids, 20or more amino acids, 30 or more amino acids, 40 or more amino acids, 50or more amino acids, 100 or more amino acids, 200 or more amino acids,300 or more amino acids, 400 or more amino acids, or 500 or more aminoacids from the reference Clostridial toxin on which the non-conservativeClostridial toxin variant is based. A non-conservative Clostridial toxinvariant can also substitute at least 10 contiguous amino acids, at least15 contiguous amino acids, at least 20 contiguous amino acids, or atleast 25 contiguous amino acids from the reference Clostridial toxin onwhich the non-conservative Clostridial toxin variant is based, thatpossess at least 50% amino acid identity, 65% amino acid identity, 75%amino acid identity, 85% amino acid identity or 95% amino acid identityto the reference Clostridial toxin on which the non-conservativeClostridial toxin variant is based. Non-limiting examples of anon-conservative Clostridial toxin variant include, e.g.,non-conservative BoNT/A variants, non-conservative BoNT/B variants,non-conservative BoNT/C1 variants, non-conservative BoNT/D variants,non-conservative BoNT/E variants, non-conservative BoNT/F variants,non-conservative BoNT/G variants, non-conservative TeNT variants,non-conservative BaNT variants and non-conservative BuNT variants.

As used herein, the term “Clostridial toxin chimeric variant” means amolecule comprising at least a portion of a Clostridial toxin and atleast a portion of at least one other protein to form a toxin with atleast one property different from the reference Clostridial toxins ofTable 1. One class of Clostridial toxin chimeric variant comprises amodified Clostridial toxin were the endogenous cell binding domain of anaturally-occurring Clostridial toxin is either modified or replacedwith a cell binding domain of another molecule. Such modifiedClostridial toxin possesses an altered cell binding activity because themodified toxin can, e.g., use the same receptor present on the surfaceof a naturally occurring Clostridial toxin target cell, referred to asan enhanced cell binding activity for a naturally-occurring Clostridialtoxin target cell; use a different receptor present on the surface of anaturally occurring Clostridial toxin target cell, referred to as analtered cell binding activity for a naturally-occurring Clostridialtoxin target cell, or use a different receptor present on the surface ofthe non-Clostridial toxin target cell, referred to as an altered cellbinding activity for a non-naturally-occurring Clostridial toxin targetcell.

A Clostridial toxin chimeric variant can be a modified Clostridial toxinwith an enhanced cell binding activity capable of intoxicating anaturally occurring Clostridial toxin target cell, e.g., a motor neuron.One way this enhanced binding activity is achieved by modifying theendogenous targeting domain of a naturally-occurring Clostridial toxinin order to enhance a cell binding activity of the toxin for itsnaturally-occurring receptor. Such modifications to a targeting domainresult in, e.g., a enhanced cell binding activity that increases bindingaffinity for an endogenous Clostridial toxin receptor present on anaturally-occurring Clostridial toxin target cell; an enhanced cellbinding activity that increases binding specificity for a subgroup ofendogenous Clostridial toxin receptors present on a naturally-occurringClostridial toxin target cell; or an enhanced cell binding activity thatincreases both binding affinity and binding specificity. Non-limitingexamples of modified Clostridial toxins an enhanced cell bindingactivity for a naturally-occurring Clostridial toxin receptor aredescribed in, e.g., Lance E. Steward, et al., Modified ClostridialToxins with Enhanced Targeting Capabilities For Endogenous ClostridialToxin Receptors, International Patent Publication No. 2006/008956 (Mar.14, 2006), Lance E. Steward, Modified Clostridial Toxins with EnhancedTranslocation Capability, and Enhanced Targeting Activity, U.S.Provisional Patent Application No. 60/807,063 (Jul. 11, 2006); thecontent of which are all hereby incorporated by reference in theirentirety.

A Clostridial toxin chimeric variant can be a modified Clostridial toxinwith an altered cell binding activity capable of intoxicating anaturally occurring Clostridial toxin target cell, e.g., a motor neuron.One way this altered capability is achieved by replacing the endogenoustargeting domain of a naturally-occurring Clostridial toxin with atargeting domain of another molecule that selectively binds to adifferent receptor present on the surface of a naturally occurringClostridial toxin target cell. Such a modification to a targeting domainresults in a modified toxin that is able to selectively bind to anon-Clostridial toxin receptor (target receptor) present on aClostridial toxin target cell. This enhanced binding activity for anaturally occurring Clostridial toxin target cell allows for lowereffective doses of a modified Clostridial toxin to be administered to anindividual because more toxin will be delivered to the target cell.Thus, modified Clostridial toxins with an enhanced binding activity willreduce the undesirable dispersal of the toxin to areas not targeted fortreatment, thereby reducing or preventing the undesirable side-effectsassociated with diffusion of a Clostridial toxin to an unwantedlocation. Non-limiting examples of modified Clostridial toxins with analtered cell binding capability for a Clostridial toxin target cell aredescribed in, e.g., Lance E. Steward et al., Modified Clostridial Toxinswith Altered Targeting Capabilities For Clostridial Toxin Target Cells,International Patent Publication No. 2006/009831 (Mar. 14, 2005); LanceE. Steward et al., Multivalent Clostridial Toxin Derivatives and Methodsof Their Use, U.S. patent application Ser. No. 11/376,696 (Mar. 15,2006); and Lance E. Steward, Modified Clostridial Toxins with EnhancedTranslocation Capabilities and Altered Targeting Activity forClostridial Toxin Target Cells, U.S. Provisional Patent Application No.60/807,062, (Jul. 11, 2006); the contents of all of which are herebyincorporated by reference in their entirety.

A Clostridial toxin chimeric variant can be a modified Clostridial toxinwith an altered cell binding activity capable of intoxicating a cellother than a naturally occurring Clostridial toxin target cell, e.g., acell other than a motor neuron. These modified toxins achieve thisintoxication by using a target receptor present on non-Clostridial toxintarget cell. This re-targeted capability is achieved by replacing anaturally-occurring targeting domain of a Clostridial toxin with atargeting domain showing a selective binding activity for anon-Clostridial toxin receptor present in a non-Clostridial toxin targetcell. Such modifications to a targeting domain result in a modifiedtoxin that is able to selectively bind to a non-Clostridial toxinreceptor (target receptor) present on a non-Clostridial toxin targetcell (re-targeted). A modified Clostridial toxin with an alteredtargeting activity for a non-Clostridial toxin target cell can bind to atarget receptor, translocate into the cytoplasm, and exert itsproteolytic effect on the SNARE complex of the non-Clostridial toxintarget cell. Non-limiting examples of modified Clostridial toxins withan altered targeting activity for a non-Clostridial toxin target cellare described in, e.g., Keith A. Foster et al., Clostridial ToxinDerivatives Able To Modify Peripheral Sensory Afferent Functions, U.S.Pat. No. 5,989,545 (Nov. 23, 1999); Clifford C. Shone et al.,Recombinant Toxin Fragments, U.S. Pat. No. 6,461,617 (Oct. 8, 2002);Conrad P. Quinn et al., Methods and Compounds for the Treatment of MucusHypersecretion, U.S. Pat. No. 6,632,440 (Oct. 14, 2003); Lance E.Steward et al., Methods And Compositions For The Treatment OfPancreatitis, U.S. Pat. No. 6,843,998 (Jan. 18, 2005); Stephan Donovan,Clostridial Toxin Derivatives and Methods For Treating Pain, U.S. PatentPublication 2002/0037833 (Mar. 28, 2002); Keith A. Foster et al.,Inhibition of Secretion from Non-neural Cells, U.S. Patent Publication2003/0180289 (Sep. 25, 2003); J. Oliver Dolly et al., ActivatableRecombinant Neurotoxins, International Patent Publication WO 2001/014570(Mar. 1, 2001); Keith A. Foster et al., Re-targeted Toxin Conjugates,International Patent Publication WO 2005/023309 (Mar. 17, 2005); LanceE. Steward et al., Multivalent Clostridial Toxin Derivatives and Methodsof Their Use, U.S. patent application Ser. No. 11/376,696 (Mar. 15,2006); Keith A. Foster, Fusion Proteins, International PatentPublication WO 2006/059093 (Jun. 8, 2005); Keith A. Foster,Non-Cytotoxic Protein Conjugates, International Patent Publication WO2006/059105 (Jun. 8, 2005); and Lance E. Steward, Modified ClostridialToxins with Enhanced Translocation Capabilities and Altered TargetingCapabilities for Non-Clostridial Toxin Target Cells, U.S. ProvisionalPatent Application No. 60/807,059, (Jul. 11, 2006); the contents of allof which are hereby incorporated by reference in their entirety. Theability to re-target the therapeutic effects associated with Clostridialtoxins has greatly extended the number of medicinal applications able touse a Clostridial toxin therapy. As a non-limiting example, modifiedClostridial toxins retargeted to sensory neurons are useful in treatingvarious kinds of chronic pain, such as, e.g., hyperalgesia andallodynia, neuropathic pain and inflammatory pain, see, e.g., Foster,supra, (1999); and Donovan, supra, (2002); and Stephan Donovan, MethodFor Treating Neurogenic Inflammation Pain with Botulinum Toxin andSubstance P Components, U.S. Pat. No. 7,022,329 (Apr. 4, 2006). Asanother non-limiting example, modified Clostridial toxins retargeted topancreatic cells are useful in treating pancreatitis, see, e.g.,Steward, supra, (2005).

Thus, in an embodiment, a Clostridial toxin chimeric variant cancomprise a modified Clostridial toxin disclosed in the presentspecification where the binding domain comprises an enhanced cellbinding activity capable of intoxicating a naturally occurringClostridial toxin target cell. In another embodiment, a Clostridialtoxin chimeric variant can comprise a modified Clostridial toxindisclosed in the present specification where the binding domaincomprises an altered cell binding activity capable of intoxicating anaturally occurring Clostridial toxin target cell. In still anotherembodiment, a Clostridial toxin chimeric variant can comprise a modifiedClostridial toxin disclosed in the present specification where thebinding domain comprises an altered cell binding activity capable ofintoxicating a non-naturally occurring Clostridial toxin target cell.

It is also envisioned that any of a variety of Clostridial toxinfragments can be useful in aspects of the present invention with theproviso that these active fragments can execute the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate. Thus, aspects of this embodiment can include Clostridialtoxin fragments having a length of, e.g., at least 300 amino acids, atleast 400 amino acids, at least 500 amino acids, at least 600 aminoacids, at least 700 amino acids, at least 800 amino acids, at least 900amino acids, at least 1000 amino acids, at least 1100 amino acids and atleast 1200 amino acids. Other aspects of this embodiment, can includeClostridial toxin fragments having a length of, e.g., at most 300 aminoacids, at most 400 amino acids, at most 500 amino acids, at most 600amino acids, at most 700 amino acids, at most 800 amino acids, at most900 amino acids, at most 1000 amino acids, at most 1100 amino acids andat most 1200 amino acids.

It is also envisioned that any of a variety of Clostridial toxinfragments comprising the light chain can be useful in aspects of thepresent invention with the proviso that these light chain fragments canspecifically target the core components of the neurotransmitter releaseapparatus and thus participate in executing the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate. The light chains of Clostridial toxins are approximately420-460 amino acids in length and comprise an enzymatic domain (Table1). Research has shown that the entire length of a Clostridial toxinlight chain is not necessary for the enzymatic activity of the enzymaticdomain. As a non-limiting example, the first eight amino acids of theBoNT/A light chain (residues 1-8 of SEQ ID NO: 1) are not required forenzymatic activity. As another non-limiting example, the first eightamino acids of the TeNT light chain (residues 1-8 of SEQ ID NO: 8) arenot required for enzymatic activity. Likewise, the carboxyl-terminus ofthe light chain is not necessary for activity. As a non-limitingexample, the last 32 amino acids of the BoNT/A light chain (residues417-448 of SEQ ID NO: 1) are not required for enzymatic activity. Asanother non-limiting example, the last 31 amino acids of the TeNT lightchain (residues 427-457 of SEQ ID NO: 8) are not required for enzymaticactivity. Thus, aspects of this embodiment can include Clostridial toxinlight chains comprising an enzymatic domain having a length of, e.g., atleast 350 amino acids, at least 375 amino acids, at least 400 aminoacids, at least 425 amino acids and at least 450 amino acids. Otheraspects of this embodiment can include Clostridial toxin light chainscomprising an enzymatic domain having a length of, e.g., at most 350amino acids, at most 375 amino acids, at most 400 amino acids, at most425 amino acids and at most 450 amino acids.

It is also envisioned that any of a variety of Clostridial toxin H_(N)regions comprising a translocation domain can be useful in aspects ofthe present invention with the proviso that these active fragments canfacilitate the release of the LC from intracellular vesicles into thecytoplasm of the target cell and thus participate in executing theoverall cellular mechanism whereby a Clostridial toxin proteolyticallycleaves a substrate. The H_(N) regions from the heavy chains ofClostridial toxins are approximately 410-430 amino acids in length andcomprise a translocation domain (Table 1). Research has shown that theentire length of a H_(N) region from a Clostridial toxin heavy chain isnot necessary for the translocating activity of the translocationdomain. Thus, aspects of this embodiment can include Clostridial toxinH_(N) regions comprising a translocation domain having a length of,e.g., at least 350 amino acids, at least 375 amino acids, at least 400amino acids and at least 425 amino acids. Other aspects of thisembodiment can include Clostridial toxin H_(N) regions comprisingtranslocation domain having a length of, e.g., at most 350 amino acids,at most 375 amino acids, at most 400 amino acids and at most 425 aminoacids.

It is also envisioned that any of a variety of Clostridial toxin H_(C)regions comprising a binding domain can be useful in aspects of thepresent invention with the proviso that these active fragments candetermine the binding activity and binding specificity of the toxin tothe receptor complex located at the surface of the target cell executethe overall cellular mechanism whereby a Clostridial toxinproteolytically cleaves a substrate. The H_(C) regions from the heavychains of Clostridial toxins are approximately 400-440 amino acids inlength and comprise a binding domain (Table 1). Research has shown thatthe entire length of a H_(C) region from a Clostridial toxin heavy chainis not necessary for the binding activity of the binding domain. Thus,aspects of this embodiment can include Clostridial toxin H_(C) regionscomprising a binding domain having a length of, e.g., at least 350 aminoacids, at least 375 amino acids, at least 400 amino acids and at least425 amino acids. Other aspects of this embodiment can includeClostridial toxin H_(C) regions comprising a binding domain having alength of, e.g., at most 350 amino acids, at most 375 amino acids, atmost 400 amino acids and at most 425 amino acids.

Thus, in an embodiment, a Clostridial toxin comprises a Clostridialtoxin enzymatic domain, a Clostridial toxin translocation domain and aClostridial toxin binding domain. In an aspect of this embodiment, aClostridial toxin comprises a naturally occurring Clostridial toxinvariant, such as, e.g., a Clostridial toxin isoform or a Clostridialtoxin subtype. In another aspect of this embodiment, a Clostridial toxincomprises a non-naturally occurring Clostridial toxin variant, such as,e.g., a conservative Clostridial toxin variant, a non-conservativeClostridial toxin variant or an active Clostridial toxin fragment, orany combination thereof. In another aspect of this embodiment, aClostridial toxin comprises a Clostridial toxin enzymatic domain or anactive fragment thereof, a Clostridial toxin translocation domain or anactive fragment thereof, a Clostridial toxin binding domain or an activefragment thereof, or any combination thereof. In other aspects of thisembodiment, a Clostridial toxin can comprise a BoNT/A, a BoNT/B, aBoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT or aBuNT.

In another embodiment, a Clostridial toxin comprises a BoNT/A. In anaspect of this embodiment, a BoNT/A comprises a BoNT/A enzymatic domain,a BoNT/A translocation domain and a BoNT/A binding domain. In anotheraspect of this embodiment, a BoNT/A comprises SEQ ID NO: 1. In anotheraspect of this embodiment, a BoNT/A comprises a naturally occurringBoNT/A variant, such as, e.g., a BoNT/A isoform or a BoNT/A subtype. Inanother aspect of this embodiment, a BoNT/A comprises a naturallyoccurring BoNT/A variant of SEQ ID NO: 1, such as, e.g., a BoNT/Aisoform of SEQ ID NO: 1 or a BoNT/A subtype of SEQ ID NO: 1. In stillanother aspect of this embodiment, a BoNT/A comprises a non-naturallyoccurring BoNT/A variant, such as, e.g., a conservative BoNT/A variant,a non-conservative BoNT/A variant or an active BoNT/A fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/A comprises a non-naturally occurring BoNT/A variant of SEQ ID NO:1, such as, e.g., a conservative BoNT/A variant of SEQ ID NO: 1, anon-conservative BoNT/A variant of SEQ ID NO: 1 or an active BoNT/Afragment of SEQ ID NO: 1, or any combination thereof. In yet anotheraspect of this embodiment, a BoNT/A comprises a BoNT/A enzymatic domainor an active fragment thereof, a BoNT/A translocation domain or anactive fragment thereof, a BoNT/A binding domain or an active fragmentthereof, or any combination thereof. In yet another aspect of thisembodiment, a BoNT/A comprising a BoNT/A enzymatic domain of amino acids1-448 from SEQ ID NO: 1 or an active fragment thereof, a BoNT/Atranslocation domain of amino acids 449-871 from SEQ ID NO: 1 or anactive fragment thereof, a BoNT/A binding domain of amino acids 872-1296from SEQ ID NO: 1 or an active fragment thereof, and any combinationthereof.

In other aspects of this embodiment, a BoNT/A comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 1, atleast 75% amino acid identity with the SEQ ID NO: 1, at least 80% aminoacid identity with SEQ ID NO: 1, at least 85% amino acid identity withSEQ ID NO: 1, at least 90% amino acid identity with SEQ ID NO: 1 or atleast 95% amino acid identity with SEQ ID NO: 1. In yet other aspects ofthis embodiment, a BoNT/A comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 1, at most 75% amino acididentity with the SEQ ID NO: 1, at most 80% amino acid identity with SEQID NO: 1, at most 85% amino acid identity with SEQ ID NO: 1, at most 90%amino acid identity with SEQ ID NO: 1 or at most 95% amino acid identitywith SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 1. In yet other aspects of this embodiment, a BoNT/A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 1. In still other aspects of this embodiment, a BoNT/A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 1.

In other aspects of this embodiment, a BoNT/A comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 1. In yet other aspects of this embodiment, a BoNT/A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ IDNO: 1. In still other aspects of this embodiment, a BoNT/A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:1.

In another embodiment, a Clostridial toxin comprises a BoNT/B. In anaspect of this embodiment, a BoNT/B comprises a BoNT/B enzymatic domain,a BoNT/B translocation domain and a BoNT/B binding domain. In anotheraspect of this embodiment, a BoNT/B comprises SEQ ID NO: 2. In anotheraspect of this embodiment, a BoNT/B comprises a naturally occurringBoNT/B variant, such as, e.g., a BoNT/β isoform or a BoNT/B subtype. Inanother aspect of this embodiment, a BoNT/B comprises a naturallyoccurring BoNT/B variant of SEQ ID NO: 2, such as, e.g., a BoNT/βisoform of SEQ ID NO: 2 or a BoNT/B subtype of SEQ ID NO: 2. In stillanother aspect of this embodiment, a BoNT/B comprises a non-naturallyoccurring BoNT/B variant, such as, e.g., a conservative BoNT/B variant,a non-conservative BoNT/B variant or an active BoNT/B fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/B comprises a non-naturally occurring BoNT/B variant of SEQ ID NO:2, such as, e.g., a conservative BoNT/B variant of SEQ ID NO: 2, anon-conservative BoNT/B variant of SEQ ID NO: 2 or an active BoNT/Bfragment of SEQ ID NO: 2, or any combination thereof. In yet anotheraspect of this embodiment, a BoNT/B comprising a BoNT/B enzymatic domainor an active fragment thereof, a BoNT/B translocation domain or activefragment thereof, a BoNT/B binding domain or active fragment thereof,and any combination thereof. In yet another aspect of this embodiment, aBoNT/B comprising a BoNT/B enzymatic domain of amino acids 1-441 fromSEQ ID NO: 2 or active fragment thereof, a BoNT/B translocation domainof amino acids 442-858 from SEQ ID NO: 2 or active fragment thereof, aBoNT/B binding domain of amino acids 859-1291 from SEQ ID NO: 2 oractive fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BoNT/B comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 2, atleast 75% amino acid identity with the SEQ ID NO: 2, at least 80% aminoacid identity with SEQ ID NO: 2, at least 85% amino acid identity withSEQ ID NO: 2, at least 90% amino acid identity with SEQ ID NO: 2 or atleast 95% amino acid identity with SEQ ID NO: 2. In yet other aspects ofthis embodiment, a BoNT/B comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 2, at most 75% amino acididentity with the SEQ ID NO: 2, at most 80% amino acid identity with SEQID NO: 2, at most 85% amino acid identity with SEQ ID NO: 2, at most 90%amino acid identity with SEQ ID NO: 2 or at most 95% amino acid identitywith SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 2. In yet other aspects of this embodiment, a BoNT/B comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 2. In still other aspects of this embodiment, a BoNT/B comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 2.

In other aspects of this embodiment, a BoNT/B comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 2. In yet other aspects of this embodiment, a BoNT/B comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO:2. In still other aspects of this embodiment, a BoNT/B comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:2.

In another embodiment, a Clostridial toxin comprises a BoNT/C1. In anaspect of this embodiment, a BoNT/C1 comprises a BoNT/C1 enzymaticdomain, a BoNT/C1 translocation domain and a BoNT/C1 binding domain. Inanother aspect of this embodiment, a BoNT/C1 comprises SEQ ID NO: 3. Inanother aspect of this embodiment, a BoNT/C1 comprises a naturallyoccurring BoNT/C1 variant, such as, e.g., a BoNT/C1 isoform or a BoNT/C1subtype. In another aspect of this embodiment, a BoNT/C1 comprises anaturally occurring BoNT/C1 variant of SEQ ID NO: 3, such as, e.g., aBoNT/C1 isoform of SEQ ID NO: 3 or a BoNT/C1 subtype of SEQ ID NO: 3. Instill another aspect of this embodiment, a BoNT/C1 comprises anon-naturally occurring BoNT/C1 variant, such as, e.g., a conservativeBoNT/C1 variant, a non-conservative BoNT/C1 variant or an active BoNT/C1fragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/C1 comprises a non-naturally occurring BoNT/C1variant of SEQ ID NO: 3, such as, e.g., a conservative BoNT/C1 variantof SEQ ID NO: 3, a non-conservative BoNT/C1 variant of SEQ ID NO: 3 oran active BoNT/C1 fragment of SEQ ID NO: 3, or any combination thereof.In yet another aspect of this embodiment, a BoNT/C1 comprises a BoNT/C1enzymatic domain or active fragment thereof, a BoNT/C1 translocationdomain or active fragment thereof, a BoNT/C1 binding domain or activefragment thereof, and any combination thereof. In yet another aspect ofthis embodiment, a BoNT/C1 comprises a BoNT/C1 enzymatic domain of aminoacid 1-449 from SEQ ID NO: 3 or active fragment thereof, a BoNT/C1translocation domain of amino acids 450-866 from SEQ ID NO: 3 or activefragment thereof, a BoNT/C1 binding domain of amino acids 867-1291 fromSEQ ID NO: 3 or active fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BoNT/C1 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 3, atleast 75% amino acid identity with the SEQ ID NO: 3, at least 80% aminoacid identity with SEQ ID NO: 3, at least 85% amino acid identity withSEQ ID NO: 3, at least 90% amino acid identity with SEQ ID NO: 3 or atleast 95% amino acid identity with SEQ ID NO: 3. In yet other aspects ofthis embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 3, at most 75% amino acididentity with the SEQ ID NO: 3, at most 80% amino acid identity with SEQID NO: 3, at most 85% amino acid identity with SEQ ID NO: 3, at most 90%amino acid identity with SEQ ID NO: 3 or at most 95% amino acid identitywith SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 3. In other aspects of thisembodiment, a BoNT/C1 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100, 200 or 500 non-contiguous amino acid substitutions relative toSEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1comprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletionsrelative to SEQ ID NO: 3. In still other aspects of this embodiment, aBoNT/C1 comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additionsrelative to SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 3. In other aspects of thisembodiment, a BoNT/C1 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQID NO: 3. In yet other aspects of this embodiment, a BoNT/C1 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 3. In other aspects of thisembodiment, a BoNT/C1 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ IDNO: 3. In still other aspects of this embodiment, a BoNT/C1 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 3. In other aspects of thisembodiment, a BoNT/C1 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100, 200 or 500 contiguous amino acid additions relative to SEQ IDNO: 3.

In another embodiment, a Clostridial toxin comprises a BoNT/D. In anaspect of this embodiment, a BoNT/D comprises a BoNT/D enzymatic domain,a BoNT/D translocation domain and a BoNT/D binding domain. In anotheraspect of this embodiment, a BoNT/D comprises SEQ ID NO: 4. In anotheraspect of this embodiment, a BoNT/D comprises a naturally occurringBoNT/D variant, such as, e.g., a BoNT/D isoform or a BoNT/D subtype. Inanother aspect of this embodiment, a BoNT/D comprises a naturallyoccurring BoNT/D variant of SEQ ID NO: 4, such as, e.g., a BoNT/Disoform of SEQ ID NO: 4 or a BoNT/D subtype of SEQ ID NO: 4. In stillanother aspect of this embodiment, a BoNT/D comprises a non-naturallyoccurring BoNT/D variant, such as, e.g., a conservative BoNT/D variant,a non-conservative BoNT/D variant or an active BoNT/D fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/D comprises a non-naturally occurring BoNT/D variant of SEQ ID NO:4, such as, e.g., a conservative BoNT/D variant of SEQ ID NO: 4, anon-conservative BoNT/D variant of SEQ ID NO: 4 or an active BoNT/Dfragment of SEQ ID NO: 4, or any combination thereof. In yet anotheraspect of this embodiment, a BoNT/D comprises a BoNT/D enzymatic domainor an active fragment thereof, a BoNT/D translocation domain or anactive fragment thereof, a BoNT/D binding domain or an active fragmentthereof, or any combination thereof. In yet another aspect of thisembodiment, a BoNT/D comprising a BoNT/D enzymatic domain of amino acids1-445 from SEQ ID NO: 4 or an active fragment thereof, a BoNT/Dtranslocation domain of amino acids 446-862 from SEQ ID NO: 4 or anactive fragment thereof, a BoNT/D binding domain of amino acids 863-1276from SEQ ID NO: 4 or an active fragment thereof, and any combinationthereof.

In other aspects of this embodiment, a BoNT/D comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 4, atleast 75% amino acid identity with the SEQ ID NO: 4, at least 80% aminoacid identity with SEQ ID NO: 4, at least 85% amino acid identity withSEQ ID NO: 4, at least 90% amino acid identity with SEQ ID NO: 4 or atleast 95% amino acid identity with SEQ ID NO: 4. In yet other aspects ofthis embodiment, a BoNT/D comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 4, at most 75% amino acididentity with the SEQ ID NO: 4, at most 80% amino acid identity with SEQID NO: 4, at most 85% amino acid identity with SEQ ID NO: 4, at most 90%amino acid identity with SEQ ID NO: 4 or at most 95% amino acid identitywith SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 4. In yet other aspects of this embodiment, a BoNT/D comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 4. In still other aspects of this embodiment, a BoNT/D comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 4.

In other aspects of this embodiment, a BoNT/D comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 4. In yet other aspects of this embodiment, a BoNT/D comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO:4. In still other aspects of this embodiment, a BoNT/D comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:4.

In another embodiment, a Clostridial toxin comprises a BoNT/E. In anaspect of this embodiment, a BoNT/E comprises a BoNT/E enzymatic domain,a BoNT/E translocation domain and a BoNT/E binding domain. In anotheraspect of this embodiment, a BoNT/E comprises SEQ ID NO: 5. In anotheraspect of this embodiment, a BoNT/E comprises a naturally occurringBoNT/E variant, such as, e.g., a BoNT/E isoform or a BoNT/E subtype. Inanother aspect of this embodiment, a BoNT/E comprises a naturallyoccurring BoNT/E variant of SEQ ID NO: 5, such as, e.g., a BoNT/Eisoform of SEQ ID NO: 5 or a BoNT/E subtype of SEQ ID NO: 5. In stillanother aspect of this embodiment, a BoNT/E comprises a non-naturallyoccurring BoNT/E variant, such as, e.g., a conservative BoNT/E variant,a non-conservative BoNT/E variant or an active BoNT/E fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/E comprises a non-naturally occurring BoNT/E variant of SEQ ID NO:5, such as, e.g., a conservative BoNT/E variant of SEQ ID NO: 5, anon-conservative BoNT/E variant of SEQ ID NO: 5 or an active BoNT/Efragment of SEQ ID NO: 5, or any combination thereof. In yet anotheraspect of this embodiment, a BoNT/E comprising a BoNT/E enzymatic domainor an active fragment thereof, a BoNT/E translocation domain or activefragment thereof, a BoNT/E binding domain or active fragment thereof,and any combination thereof. In yet another aspect of this embodiment, aBoNT/E comprising a BoNT/E enzymatic domain of amino acids 1-422 fromSEQ ID NO: 5 or active fragment thereof, a BoNT/E translocation domainof amino acids 423-845 from SEQ ID NO: 5 or active fragment thereof, aBoNT/E binding domain of amino acids 846-1252 from SEQ ID NO: 5 oractive fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BoNT/E comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 5, atleast 75% amino acid identity with the SEQ ID NO: 5, at least 80% aminoacid identity with SEQ ID NO: 5, at least 85% amino acid identity withSEQ ID NO: 5, at least 90% amino acid identity with SEQ ID NO: 5 or atleast 95% amino acid identity with SEQ ID NO: 5. In yet other aspects ofthis embodiment, a BoNT/E comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 5, at most 75% amino acididentity with the SEQ ID NO: 5, at most 80% amino acid identity with SEQID NO: 5, at most 85% amino acid identity with SEQ ID NO: 5, at most 90%amino acid identity with SEQ ID NO: 5 or at most 95% amino acid identitywith SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 5. In yet other aspects of this embodiment, a BoNT/E comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 5. In still other aspects of this embodiment, a BoNT/E comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 5.

In other aspects of this embodiment, a BoNT/E comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 5. In yet other aspects of this embodiment, a BoNT/E comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO:5. In still other aspects of this embodiment, a BoNT/E comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:5.

In another embodiment, a Clostridial toxin comprises a BoNT/F. In anaspect of this embodiment, a BoNT/F comprises a BoNT/F enzymatic domain,a BoNT/F translocation domain and a BoNT/F binding domain. In anotheraspect of this embodiment, a BoNT/F comprises SEQ ID NO: 6. In anotheraspect of this embodiment, a BoNT/F comprises a naturally occurringBoNT/F variant, such as, e.g., a BoNT/F isoform or a BoNT/F subtype. Inanother aspect of this embodiment, a BoNT/F comprises a naturallyoccurring BoNT/F variant of SEQ ID NO: 6, such as, e.g., a BoNT/Fisoform of SEQ ID NO: 6 or a BoNT/F subtype of SEQ ID NO: 6. In stillanother aspect of this embodiment, a BoNT/F comprises a non-naturallyoccurring BoNT/F variant, such as, e.g., a conservative BoNT/F variant,a non-conservative BoNT/F variant or an active BoNT/F fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/F comprises a non-naturally occurring BoNT/F variant of SEQ ID NO:6, such as, e.g., a conservative BoNT/F variant of SEQ ID NO: 6, anon-conservative BoNT/F variant of SEQ ID NO: 6 or an active BoNT/Ffragment of SEQ ID NO: 6, or any combination thereof. In yet anotheraspect of this embodiment, a BoNT/F comprises a BoNT/F enzymatic domainor active fragment thereof, a BoNT/F translocation domain or activefragment thereof, a BoNT/F binding domain or active fragment thereof,and any combination thereof. In yet another aspect of this embodiment, aBoNT/F comprises a BoNT/F enzymatic domain of amino acid 1-439 from SEQID NO: 6 or active fragment thereof, a BoNT/F translocation domain ofamino acids 440-864 from SEQ ID NO: 6 or active fragment thereof, aBoNT/F binding domain of amino acids 865-1274 from SEQ ID NO: 6 oractive fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BoNT/F comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 6, atleast 75% amino acid identity with the SEQ ID NO: 6, at least 80% aminoacid identity with SEQ ID NO: 6, at least 85% amino acid identity withSEQ ID NO: 6, at least 90% amino acid identity with SEQ ID NO: 6 or atleast 95% amino acid identity with SEQ ID NO: 6. In yet other aspects ofthis embodiment, a BoNT/F comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 6, at most 75% amino acididentity with the SEQ ID NO: 6, at most 80% amino acid identity with SEQID NO: 6, at most 85% amino acid identity with SEQ ID NO: 6, at most 90%amino acid identity with SEQ ID NO: 6 or at most 95% amino acid identitywith SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 6. In yet other aspects of this embodiment, a BoNT/F comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 6. In still other aspects of this embodiment, a BoNT/F comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 6.

In other aspects of this embodiment, a BoNT/F comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 6. In yet other aspects of this embodiment, a BoNT/F comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO:6. In still other aspects of this embodiment, a BoNT/F comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:6.

In another embodiment, a Clostridial toxin comprises a BoNT/G. In anaspect of this embodiment, a BoNT/G comprises a BoNT/G enzymatic domain,a BoNT/G translocation domain and a BoNT/G binding domain. In anotheraspect of this embodiment, a BoNT/G comprises SEQ ID NO: 7. In anotheraspect of this embodiment, a BoNT/G comprises a naturally occurringBoNT/G variant, such as, e.g., a BoNT/G isoform or a BoNT/G subtype. Inanother aspect of this embodiment, a BoNT/G comprises a naturallyoccurring BoNT/G variant of SEQ ID NO: 7, such as, e.g., a BoNT/Gisoform of SEQ ID NO: 7 or a BoNT/G subtype of SEQ ID NO: 7. In stillanother aspect of this embodiment, a BoNT/G comprises a non-naturallyoccurring BoNT/G variant, such as, e.g., a conservative BoNT/G variant,a non-conservative BoNT/G variant or an active BoNT/G fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/D comprises a non-naturally occurring BoNT/G variant of SEQ ID NO:7, such as, e.g., a conservative BoNT/G variant of SEQ ID NO: 7, anon-conservative BoNT/G variant of SEQ ID NO: 7 or an active BoNT/Gfragment of SEQ ID NO: 7, or any combination thereof. In yet anotheraspect of this embodiment, a BoNT/G comprises a BoNT/G enzymatic domainor an active fragment thereof, a BoNT/G translocation domain or anactive fragment thereof, a BoNT/G binding domain or an active fragmentthereof, or any combination thereof. In yet another aspect of thisembodiment, a BoNT/G comprising a BoNT/G enzymatic domain of amino acids1-446 from SEQ ID NO: 7 or an active fragment thereof, a BoNT/Gtranslocation domain of amino acids 447-863 from SEQ ID NO: 7 or anactive fragment thereof, a BoNT/G binding domain of amino acids 864-1297from SEQ ID NO: 7 or an active fragment thereof, and any combinationthereof.

In other aspects of this embodiment, a BoNT/G comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 7, atleast 75% amino acid identity with the SEQ ID NO: 7, at least 80% aminoacid identity with SEQ ID NO: 7, at least 85% amino acid identity withSEQ ID NO: 7, at least 90% amino acid identity with SEQ ID NO: 7 or atleast 95% amino acid identity with SEQ ID NO: 7. In yet other aspects ofthis embodiment, a BoNT/G comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 7, at most 75% amino acididentity with the SEQ ID NO: 7, at most 80% amino acid identity with SEQID NO: 7, at most 85% amino acid identity with SEQ ID NO: 7, at most 90%amino acid identity with SEQ ID NO: 7 or at most 95% amino acid identitywith SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 7. In yet other aspects of this embodiment, a BoNT/G comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 7. In still other aspects of this embodiment, a BoNT/G comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 7.

In other aspects of this embodiment, a BoNT/G comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 7. In yet other aspects of this embodiment, a BoNT/G comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO:7. In still other aspects of this embodiment, a BoNT/G comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:7.

In another embodiment, a Clostridial toxin comprises a TeNT. In anaspect of this embodiment, a TeNT comprises a TeNT enzymatic domain, aTeNT translocation domain and a TeNT binding domain. In an aspect ofthis embodiment, a TeNT comprises SEQ ID NO: 8. In another aspect ofthis embodiment, a TeNT comprises a naturally occurring TeNT variant,such as, e.g., a TeNT isoform or a TeNT subtype. In another aspect ofthis embodiment, a TeNT comprises a naturally occurring TeNT variant ofSEQ ID NO: 8, such as, e.g., a TeNT isoform of SEQ ID NO: 8 or a TeNTsubtype of SEQ ID NO: 8. In still another aspect of this embodiment, aTeNT comprises a non-naturally occurring TeNT variant, such as, e.g., aconservative TeNT variant, a non-conservative TeNT variant or an activeTeNT fragment, or any combination thereof. In still another aspect ofthis embodiment, a TeNT comprises a non-naturally occurring TeNT variantof SEQ ID NO: 8, such as, e.g., a conservative TeNT variant of SEQ IDNO: 8, a non-conservative TeNT variant of SEQ ID NO: 8 or an active TeNTfragment of SEQ ID NO: 8, or any combination thereof. In yet anotheraspect of this embodiment, a TeNT comprising a TeNT enzymatic domain oran active fragment thereof, a TeNT translocation domain or activefragment thereof, a TeNT binding domain or active fragment thereof, andany combination thereof. In yet another aspect of this embodiment, aTeNT comprising a TeNT enzymatic domain of amino acids 1-457 from SEQ IDNO: 8 or active fragment thereof, a TeNT translocation domain of aminoacids 458-879 from SEQ ID NO: 8 or active fragment thereof, a TeNTbinding domain of amino acids 880-1315 from SEQ ID NO: 8 or activefragment thereof, and any combination thereof.

In other aspects of this embodiment, a TeNT comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 8, atleast 75% amino acid identity with the SEQ ID NO: 8, at least 80% aminoacid identity with SEQ ID NO: 8, at least 85% amino acid identity withSEQ ID NO: 8, at least 90% amino acid identity with SEQ ID NO: 8 or atleast 95% amino acid identity with SEQ ID NO: 8. In yet other aspects ofthis embodiment, a TeNT comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 8, at most 75% amino acididentity with the SEQ ID NO: 8, at most 80% amino acid identity with SEQID NO: 8, at most 85% amino acid identity with SEQ ID NO: 8, at most 90%amino acid identity with SEQ ID NO: 8 or at most 95% amino acid identitywith SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 8. In yet other aspects of this embodiment, a TeNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 8. In still other aspects of this embodiment, a TeNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 8.

In other aspects of this embodiment, a TeNT comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 8. In yet other aspects of this embodiment, a TeNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO:8. In still other aspects of this embodiment, a TeNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:8.

In another embodiment, a Clostridial toxin comprises a BaNT. In anaspect of this embodiment, a BaNT comprises a BaNT enzymatic domain, aBaNT translocation domain and a BaNT binding domain. In another aspectof this embodiment, a BaNT comprises SEQ ID NO: 9. In another aspect ofthis embodiment, a BaNT comprises a naturally occurring BaNT variant,such as, e.g., a BaNT isoform or a BaNT subtype. In another aspect ofthis embodiment, a BaNT comprises a naturally occurring BaNT variant ofSEQ ID NO: 9, such as, e.g., a BaNT isoform of SEQ ID NO: 9 or a BaNTsubtype of SEQ ID NO: 9. In still another aspect of this embodiment, aBaNT comprises a non-naturally occurring BaNT variant, such as, e.g., aconservative BaNT variant, a non-conservative BaNT variant or an activeBaNT fragment, or any combination thereof. In still another aspect ofthis embodiment, a BaNT comprises a non-naturally occurring BaNT variantof SEQ ID NO: 9, such as, e.g., a conservative BaNT variant of SEQ IDNO: 9, a non-conservative BaNT variant of SEQ ID NO: 9 or an active BaNTfragment of SEQ ID NO: 9, or any combination thereof. In yet anotheraspect of this embodiment, a BaNT comprises a BaNT enzymatic domain oran active fragment thereof, a BaNT translocation domain or an activefragment thereof, a BaNT binding domain or an active fragment thereof,or any combination thereof. In yet another aspect of this embodiment, aBaNT comprising a BaNT enzymatic domain of amino acids 1-448 from SEQ IDNO: 9 or an active fragment thereof, a BaNT translocation domain ofamino acids 449-871 from SEQ ID NO: 9 or an active fragment thereof, aBaNT binding domain of amino acids 872-1296 from SEQ ID NO: 9 or anactive fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BaNT comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 9, atleast 75% amino acid identity with the SEQ ID NO: 9, at least 80% aminoacid identity with SEQ ID NO: 9, at least 85% amino acid identity withSEQ ID NO: 9, at least 90% amino acid identity with SEQ ID NO: 9 or atleast 95% amino acid identity with SEQ ID NO: 9. In yet other aspects ofthis embodiment, a BaNT comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 9, at most 75% amino acididentity with the SEQ ID NO: 9, at most 80% amino acid identity with SEQID NO: 9, at most 85% amino acid identity with SEQ ID NO: 9, at most 90%amino acid identity with SEQ ID NO: 9 or at most 95% amino acid identitywith SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 9. In other aspects of thisembodiment, a BaNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 9. In yet other aspects of this embodiment, a BaNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 9. In other aspects of thisembodiment, a BaNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 9. In still other aspects of this embodiment, a BaNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 9. In other aspects of thisembodiment, a BaNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 9.

In other aspects of this embodiment, a BaNT comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 9. In other aspects of thisembodiment, a BaNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 9. In yet other aspects of this embodiment, a BaNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 9. In other aspects of thisembodiment, a BaNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO:9. In still other aspects of this embodiment, a BaNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 9. In other aspects of thisembodiment, a BaNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:9.

In another embodiment, a Clostridial toxin comprises a BuNT. In anaspect of this embodiment, a BuNT comprises a BuNT enzymatic domain, aBuNT translocation domain and a BuNT binding domain. In another aspectof this embodiment, a BuNT comprises SEQ ID NO: 10. In another aspect ofthis embodiment, a BuNT comprises a naturally occurring BuNT variant,such as, e.g., a BuNT isoform or a BuNT subtype. In another aspect ofthis embodiment, a BuNT comprises a naturally occurring BuNT variant ofSEQ ID NO: 10, such as, e.g., a BuNT isoform of SEQ ID NO: 10 or a BuNTsubtype of SEQ ID NO: 10. In still another aspect of this embodiment, aBuNT comprises a non-naturally occurring BuNT variant, such as, e.g., aconservative BuNT variant, a non-conservative BuNT variant or an activeBuNT fragment, or any combination thereof. In still another aspect ofthis embodiment, a BuNT comprises a non-naturally occurring BuNT variantof SEQ ID NO: 10, such as, e.g., a conservative BuNT variant of SEQ IDNO: 10, a non-conservative BuNT variant of SEQ ID NO: 10 or an activeBuNT fragment of SEQ ID NO: 10, or any combination thereof. In yetanother aspect of this embodiment, a BuNT comprises a BuNT enzymaticdomain or an active fragment thereof, a BuNT translocation domain or anactive fragment thereof, a BuNT binding domain or an active fragmentthereof, or any combination thereof. In yet another aspect of thisembodiment, a BuNT comprising a BuNT enzymatic domain of amino acids1-448 from SEQ ID NO: 10 or an active fragment thereof, a BuNTtranslocation domain of amino acids 449-871 from SEQ ID NO: 10 or anactive fragment thereof, a BuNT binding domain of amino acids 872-1296from SEQ ID NO: 10 or an active fragment thereof, and any combinationthereof.

In other aspects of this embodiment, a BuNT comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 10, atleast 75% amino acid identity with the SEQ ID NO: 10, at least 80% aminoacid identity with SEQ ID NO: 10, at least 85% amino acid identity withSEQ ID NO: 10, at least 90% amino acid identity with SEQ ID NO: 10 or atleast 95% amino acid identity with SEQ ID NO: 10. In yet other aspectsof this embodiment, a BuNT comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 10, at most 75% amino acididentity with the SEQ ID NO: 10, at most 80% amino acid identity withSEQ ID NO: 10, at most 85% amino acid identity with SEQ ID NO: 10, atmost 90% amino acid identity with SEQ ID NO: 10 or at most 95% aminoacid identity with SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 10. In other aspects of thisembodiment, a BuNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 10. In yet other aspects of this embodiment, a BuNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 10. In other aspects of thisembodiment, a BuNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 10. In still other aspects of this embodiment, a BuNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 10. In other aspects of thisembodiment, a BuNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 10.

In other aspects of this embodiment, a BuNT comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 10. In other aspects of thisembodiment, a BuNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 10. In yet other aspects of this embodiment, a BuNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 10. In other aspects of thisembodiment, a BuNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO:10. In still other aspects of this embodiment, a BuNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 10. In other aspects of thisembodiment, a BuNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:10.

Using this information it is possible to construct an expressible opennucleic acid reading frame for insertion into an expression vector andsubsequent expression within a chosen host cell. Indeed, InternationalPatent publication WO 01/14570 discloses methods of making single-chain,cleavable recombinant modified or unmodified Clostridial neurotoxinderivatives and chimeric and hybrid forms thereof using such methods.Additional publications disclosing methods of making expressiblerecombinant neurotoxins and derivatives thereof include U.S. Pat. Nos.5,989,545; 6,203,794; 6,395,513; U.S. Publication Numbers U.S.2003/0166238; U.S. 2002/169942; U.S. 2004/176299; U.S. 2004/126397; U.S.2005/035730; U.S. 2005/068494; U.S. 2006/011966; International PatentApplications W095/32738; WO 99/55359; W096/33273; W098/07864;W099/17806; WO98/07864; WO02/44199; WO02/40506. All these publicationsare incorporated by reference herein in their entirety.

The use of recombinant DNA techniques permits the construction ofmodified clostridial neurotoxins having different functional propertiesfrom the naturally occurring toxin subtypes and strains thereof. Forexample, altering the naturally occurring amino acid sequence of thenative neurotoxin light chain and/or adding a different therapeuticmoiety permits the construction of transport proteins designed to carrya therapeutic agent within a neuron. See U.S. Pat. No. 6,203,794(incorporated by reference herein). Altering the targeting (binding)domain permits the toxin to be transported within pancreatic cells, suchas acinar cells, thereby preventing secretion of activated digestiveenzymes by such cells, See U.S. Pat. No. 6,843,998 (hereby incorporatedby reference herein), or sensory afferent neurons, thereby preventingneurotransmitter release and thus providing relief from pain; see U.S.Pat. No. 6,395,513 (hereby incorporated by reference herein.)

In addition, the creation of chimeric neurotoxin derivatives comprising,for example, the binding domain and the translocation domain (ormodified versions thereof) of one neurotoxin subtype for example,BoNT/A, and the light chain region of another neurotoxin subtype, forexample, BoNT/E. It will be seen that given the general structuralhomology between the neurotoxin subtypes, any combination of the threebasic clostridial neurotoxin domains, may be made in a single amino acidchain (or cleaved di-chain molecule). Thus, a binding region from any ofneurotoxin BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G,TeNT, BaNT or BuNT may be independently combined with a translocationdomain from BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G,TeNT, BaNT or BuNT, and further independently combined with aendopeptidase domain from BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E,BoNT/F, BoNT/G, TeNT, BaNT or BuNT.

The therapeutic utility of a Clostridial toxin may be enhanced oraltered by the inclusion of at least two cell binding domains. All suchmodified Clostridial toxins are referred to herein as “multivalentClostridial toxin.” Each binding domain of a multivalent Clostridialtoxin may comprise any binding domain capable of binding underphysiological conditions to a cell surface receptor capable ofselectively binding to the ligand. Furthermore, each binding domain iscapable of selectively binding to a receptor present on the surface of atarget cell, or facilitating the entry of the multivalent Clostridialtoxin into a target cell. In addition, at least one of said bindingdomains will bind selectively to the target cell and at least one of thebinding domains will bind to a cell surface receptor capable ofmediating the internalization of the multivalent Clostridial toxinwithin the target cell. By adding, for example, multiple binding domainsthe binding constant (Kd) (which equals [toxin][targetcell]/[toxin:target cell complex]) of the multivalent Clostridial toxinwill be increased over that of an unmodified toxin or monovalent toxin.

Aspects of the present invention provide, in part, a binding domain. Asused herein, the term “binding domain” is synonymous with “ligand” andmeans any molecule that can selectively interact with another moleculepresent on the surface of a cell and initiate the overallinternalization mechanism whereby the multivalent Clostridial toxindisclosed in the present specification intoxicates a target cell. Eachof the two or more binding domains in a multivalent Clostridialneurotoxin may be, for example, a binding domain that facilitatesstability, solubility and/or cell penetration. At least one such bindingdomain is selective for a cell surface molecule of a target cell. Inaddition, at least one cell surface molecule to which a binding domainof a multivalent Clostridial neurotoxin binds is capable of beinginternalized by the target cell after binding. Preferably, at least onecell surface molecule to which a binding domain of a multivalentClostridial neurotoxin selectively binds is capable of beinginternalized by the target cell after binding. As used herein, the term“selectively” means having a highly preferred activity or effect. Asused herein, the term “selectively bind” or “selective binding” means amolecule is able to bind its target receptor under physiologicalconditions, or in vitro conditions substantially approximatingphysiological conditions, to a statistically significantly greaterdegree relative to other, non-target receptors. Thus, with reference toa binding domain or ligand of the present specification, there is adiscriminatory binding of the binding domain to a cell surface receptorpresent on a cell. Thus, in an embodiment, a binding domain or ligandcan selectively bind a cell surface receptor with a dissociationconstant (Kd) of the ligand for target receptor by at least four fold,at least 10 fold, at least 50 fold, at least 100 fold, at least 1000, atleast 100,000 fold the value of Kd of the ligand for other cell surfacereceptors.

A binding domain disclosed in the present specification may facilitatethe binding activity of a multivalent Clostridial toxin to a moleculelocated at the surface of a cell. As used herein, the term “bindingactivity” means that one molecule is directly or indirectly contactinganother molecule via at least one intermolecular or intramolecularforce, including, without limitation, a covalent bond, an ionic bond, ametallic bond, a hydrogen bond, a hydrophobic interaction, a van derWaals interaction, and the like, or any combination thereof. “Bound” and“bind” are considered terms for binding.

As used herein, the term “binding affinity” means how strong a bindingdomain's activity is for a particular cell surface molecule. In general,high binding affinity results from greater intermolecular force betweena binding domain and its cell surface molecule while low bindingaffinity involves less intermolecular force between the ligand and itscell surface molecule. High binding affinity involves a longer residencetime for the binding domain at its cell surface molecule binding sitethan is the case for low binding affinity. As such, a binding domainwith a high binding affinity means a lower concentration of that bindingdomain is required to maximally occupy the binding sites of a cellsurface molecule and trigger a physiological response. Conversely, lowbinding affinity means a relatively high concentration of a bindingdomain is required before the binding sites of a cell surface moleculeis maximally occupied and the maximum physiological response isachieved. Thus, multivalent Clostridial toxins with increased bindingactivity due to high binding affinity will allow administration ofreduced doses of the toxin, thereby reducing or preventing unwantedside-effects associated with toxin dispersal into non-targeted areas.

As used herein, the term “binding specificity” means how specific abinding domain's activity is one particular cell surface molecule. Ingeneral, high binding specificity results in a more exclusiveinteraction with one particular cell surface molecule or subgroup ofcell surface molecules while low binding specificity results in a morepromiscuous interaction with a larger group of cell surface molecules.As such, a binding domain with a high binding specificity means thatbinding domain will occupy the binding sites of a particular cellsurface molecule and trigger a physiological response. Conversely, lowbinding specificity means a binding domain will occupy the binding sitesof many cell surface molecules and trigger a multitude of physiologicalresponses. Thus, multivalent Clostridial toxins with increased bindingactivity due to high binding specificity will only target cell surfacemolecules present on a subgroup of target cells, thereby reducing theside effects associated with the targeting of all target cells.

As used herein, the term “cell surface molecule” means both atraditional receptor capable of binding a ligand and thereby causing aligand-selective response (at e.g., the cell surface, the cytoplasm, orboth), and a cell surface feature such as caveolae, membrane “patches”,or lipid rafts, which appear to serve at least in part to gathertogether and locally concentrate at the cell surface specific receptorsand bound ligand types for later processing such as endocytosis, oftenmediated by a cell surface receptor.

Thus, in an embodiment, a multivalent Clostridial toxin can comprises aplurality of binding domains. In aspects of this embodiment, amultiivalent Clostridal toxion can comprise, e.g., at least two bindingdomains, at least three binding domains, at least four binding domainsor at least five binding domains. In other aspects of this embodiment, amultiivalent Clostridal toxion can comprise, e.g., at most two bindingdomains, at most three binding domains, at most four binding domains orat most five binding domains. In other aspects of this embodiment, amultiivalent Clostridal toxion comprises, e.g., two binding domains,three binding domains, four binding domains or five binding domains.

It is envisioned that any and all binding domains capable of binding aClostridial toxin receptor present on a naturally-occurring Clostridialtoxin target cell can be used to practice aspects of the presentinvention, including, without limitation, a Clostridial toxin bindingdomain, such as, e.g., a BoNT/A binding domain, a BoNT/B binding domain,a BoNT/C1 binding domain, a BoNT/D binding domain, a BoNT/E bindingdomain, a BoNT/F binding domain, a BoNT/G binding domain, a TeNT bindingdomain, a BaNT binding domain, and a BuNT binding domain; a Clostridialnon-toxin associated protein, such as, e.g., a BoNT/A HA-33, a BoNT/BHA-33, a BoNT/C1 HA-33, a BoNT/D HA-33, a BoNT/A HA-17, a BoNT/B HA-17,a BoNT/C1 HA-17, a BoNT/D HA-17, a BoNT/A NTNH, a BoNT/B NTNH, a BoNT/C1NTNH, a BoNT/D NTNH, a BoNT/E NTNH, a BoNT/F NTNH and a BoNT/G NTNH; anda FGF, such as, e.g., a FGF-1, a FGF-2, a FGF-4, a FGF-8, a FGF-9, aFGF-17 and a FGF-18.

It is envisioned that any and all binding domains capable of binding anon-Clostridial toxin receptor present on a naturally-occurringClostridial toxin target cell can be used to practice aspects of thepresent invention, including, without limitation, polypeptides thatselectively bind to a receptor present on a presynaptic membrane andpolypeptides that selectively bind to a receptor present on apostsynaptic membrane. Polypeptides that appear to bind to a receptorpresent on a presynaptic membrane, include, without limitation, Glucagonlike hormones, such as, e.g., secretin, Ghrelin (GHS), glucagon-likepeptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), pituitary adenylatecyclase activating peptide (PACAP), glicentin, glicentin-relatedpolypeptide (GRPP), oxyntomodulin (OXY), vasoactive intestinal peptide-1(VIP-1), vasoactive intestinal peptide-2 (VIP-2), gastric inhibitorypolypeptide (GIP), a galanin (Gal) or a calcitonin-relatedpeptidesvisceral gut peptide; neurohormones, such as, e.g.,corticotropin-releasing hormone (CCRH) and parathyroid hormone (PTH);neuroregulatory cytokines, such as, e.g., ciliary neurotrophic factor(CNTF), glycophorin-A (GPA), leukemia inhibitory factor (LIF), aninterleukin (IL), onostatin M (OSM), cardiotrophin-1 (CT-1),cardiotrophin-like cytokine (CLC), neuroleukin (NL), VEGF, insulin-likegrowth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2) andepidermal growth factor (EGF); neurotrophins, such as, e.g., nervegrowth factors (NGFs), brain-derived growth factors (BDNFs),neurotrophin-3s (NT-3s) and neurotrophin-4/5s (NT-4/5s); growth factors,such as, e.g., glial cell derived neurotrophic factor (GDNF), neurturin(NRTN), persephrin (PSPN), artemin (ARTN); transformation growth factorbetas (TGFβs), bone morphogenetic proteins (BMPs), growth anddifferentiation factors (GDFs), activins; axon guidance signalingmolecules, such as, e.g., netrins, semaphrorings and ephrins; sugarbinding proteins, such as, e.g., serum amyloid P, β-glucanase,sialidase, lectin, cryia, insecticidal delta-endotoxin, agglutinin,abrin and ricin; ligands that selectively bind neurexins, such as, e.g.,ligands for neurexin-1α and neurexin-1β; ligands for neurexin-2α andneurexin-2β; and ligands for neurexin-3α and neurexin-3β; and WNTs.Ligands that appear to bind to a receptor present on a postsynapticmembrane, include, without limitation, Ng-CAM (L1), NCAM, N-cadherin,Agrin-MUSK, basement membrane polypeptides, such as, e.g., laminin β-2.

It is envisioned that any and all binding domains capable of binding anon-Clostridial toxin receptor present on a non-Clostridial toxin targetcell can be used to practice aspects of the present invention,including, without limitation, polypeptides that selectively bind to areceptor present on a sensory neuron, an autonomic neuron or anon-neuronal cell. Such binding domains include, without limitation, anopioid peptide, such as, e.g., an enkephalin, a bovineadrenomedullary-22 (BAM22) peptide, an endomorphin, an endorphin, adynorphin, a nociceptin or a hemorphin; a melanocortin peptide, such as,e.g., an α-melanocyte stimulating hormones (α-MSH), a β-melanocytestimulating hormones (β-MSH), a γ-melanocyte stimulating hormones(γ-MSH), an adrenocorticotropin (ACTH), a Corticotropin-likeintermediary peptide (CLIP), a β-lipotropin (β-LPH) and a γ-lipotropin(γ-LPH); a galanin, such as, e.g., a galanin and a galaninmessage-associated peptide (GMAP); a granin, such as, e.g., achromogranin A peptide like a β-granin, a vasostatin, a chromostatin, apancreastatin, a WE-14, a catestatin, a parastatin and a GE-25, achromogranin B (secretogranin I) peptide like a GAWK peptide, anadrenomedullary peptide and a secretolytin and a chromogranin C(secretogranin II) peptide like secretoneurin, EM66 and manserin; atachykinin peptide, such as, e.g., Substance P, neuropeptide K (NPK),neuropeptide gamma (NP gamma), neurokinin A (NKA; Substance K,neurokinin alpha, neuromedin L), neurokinin B (NKB), a hemokinin and aendokinin; a cholecystokinin peptide, such as, e.g., a cholecystokinin58, a cholecystokinin 39, a cholecystokinin 33, a cholecystokinin 12 anda cholecystokinin 8; a Neuropeptide Y related peptide, such as, e.g., aNeuropeptide Y (NPY), a Peptide YY (PYY), Pancreatic peptide (PP) and aPancreatic icosapeptide (PIP); a kinin peptide, such as, e.g., abradykinin, a kallidan, a desArg⁹ bradykinin and a desArg¹⁰ bradykinin;a protease activated receptor (PAR) peptide, such as, e.g., a PAR1peptide, a PAR2 peptide, a PAR3 peptide and a PAR4 peptide; acorticotropin-releasing hormone; a thyrotropin-releasing hormone; asomatostatin; a leukemia inhibitor factor (LIF); and an interleukin-1(IL1).

It is envisioned that any and all binding domains capable offacilitating the transport of the multivalent Clostridial toxin across acell membrane of a target cell can be used to practice aspects of thepresent invention, including, without limitation, a translocator like aprotein translocation domain (PTD), such as, e.g., a herpes simplexvirus type 1 VP22 protein translocating sequence, a SV-40 virus large Ttranslocating sequence, a TAT translocating sequence, an adenovirustranslocating sequence, a synthetic integrin binding domaintranslocating sequence, a Kaposi fibroblast growth factor membranetranslocating sequence, a nuclear localization signal, a Transportantranslocating sequence, a ciliary neurotrophic factor translocatingsequence, a caveolin, an interleukin 1-β translocating sequence, athioredoxin translocating sequence, a fibroblast growth factor-1translocating sequence, a fibroblast growth factor-2 translocatingsequence, an integrin β1 translocating sequence, an integrin β3translocating sequence, a lactoferrin translocating sequence, ahomeodomain translocating sequence, like, a penetratin translocatingsequence, an Engrailed-1 translocating sequence, an Engrailed-2translocating sequence, a Hoxa-5 translocating sequence, a Hoxb-4translocating sequence, a Hoxc-8 translocating sequence

It is envisioned that any and all binding domains capable of binding toa cell surface molecule capable of facilitating the transport of amultivalent Clostridial toxin across a cell membrane of a target cellcan be used to practice aspects of the present invention, including,without limitation, an antibody to a coated pit protein, such as, e.g.,a clatherin antibody and an Adaptor Protein-2 (adaptin) antibody; anantibody to a caveolae-associated protein, such as, e.g., a caveolin-1antibody and a GPI-linked receptor protein antibody.

An example of a binding domain, includes, without limitation, aClostridial toxin binding domain such, as, e.g., a BoNT/A bindingdomain, a BoNT/B binding domain, a BoNT/C1 binding domain, a BoNT/Dbinding domain, a BoNT/E binding domain, a BoNT/F binding domain, aBoNT/G binding domain, a TeNT binding domain, a BaNT binding domain anda BuNT binding domain

The three-dimensional crystal structures of BoNT/A, BoNT/B and the H_(C)domain of TeNT indicate that the carboxyl-terminal H_(CC) domaincomprises a modified β-trefoil domain which forms three distinctcarbohydrate binding regions domains that resembles the carbohydratebinding moiety found in many sugar-binding proteins, such as, e.g.,serum amyloid P, sialidase, cryia, insecticidal ∂-endotoxin and lectins(FIG. 3). Biochemical studies indicate that the β-trefoil domainstructure of the H_(CC) domain appears to mediate the binding tospecific carbohydrate containing components of the Clostridial toxinreceptor on the cell surface, see, e.g., Krzysztof Ginalski et al.,Structure-based Sequence Alignment for the Beta-Trefoil Subdomain of theClostridial Neurotoxin Family Provides Residue Level Information Aboutthe Putative Ganglioside Binding Site, 482(1-2) FEBS Lett. 119-124(2000).

Proteins containing the structural β-trefoil domain represents a diversegroup of proteins, see, e.g., C. A. Orengo et al., Protein Superfamiliesand Domain Superfolds, 372 Nature 631-634 (1994). The β-trefoil domaincomprises a six-stranded β-barrel closed off at one end by threeβ-hairpin structures that exhibits a characteristic pseudo-threefoldaxis symmetry. The monomeric structural unit of this three-fold symmetryis referred to as the β-trefoil fold that contains four β-sheetsorganized as a pair of antiparallel β-sheets. Dividing each of theseβ-trefoil folds is a β-hairpin turn. Therefore, in a linear fashion, aβ-trefoil domain comprises four β-sheets of the first β-trefoil fold(α-fold), a β-hairpin turn, four β-sheets of the second β-trefoil fold(β-fold), a second β-hairpin turn four β-sheets of the third β-trefoilfold (γ-fold) (see FIG. 2). Because the first hairpin turn is locatedbetween the fourth and fifth β-sheets of the β-trefoil domain, it isdesignated the β4/β5 β-hairpin turn. Likewise, since the second hairpinturn is located between the eight and ninth β-sheets of the β-trefoildomain, it is designated the β8/β9 β-hairpin turn.

TABLE 2 β-trefoil Domains of Clostridial Toxins Amino Acid SequenceRegion of Carbohydrate Binding Moieties β4/β5 β8/β9 Protein SEQ ID NO:α-fold β-hairpin turn β-fold β-hairpin turn γ-fold BoNT/A 1 1111-11621163-1178 1179-1223 1224-1236 1237-1296 BoNT/B 2 1098-1147 1148-11651166-1210 1211-1222 1223-1291 BoNT/C1 3 1112-1150 1151-1166 1167-12181219-1229 1230-1291 BoNT/D 4 1099-1137 1138-1153 1154-1207 1208-12181219-1276 BoNT/E 5 1086-1129 1130-1146 1147-1190 1191-1198 1199-1252BoNT/F 6 1106-1152 1153-1171 1172-1213 1214-1221 1222-1274 BoNT/G 71106-1153 1154-1172 1173-1218 1219-1230 1231-1297 TeNT 8 1128-11771178-1194 1195-1240 1241-1254 1255-1315 BaNT 9 1095-1142 1143-11611162-1207 1208-1215 1216-1268 BuNT 10  1086-1129 1130-1146 1147-11901191-1197 1198-1251

Continuing research has elucidated that β4/β5 and β8/β9 β-hairpin turnsare important in conferring the proper pseudo-threefold axis symmetryobserved in the β-trefoil domain. Additionally, this work hasdemonstrated that amino acid changes in these two β-hairpin turns canincrease the stability of the β-trefoil domain, which in turn results inincreased binding activity, see, e.g., Stephen R. Brych et al.,Structure and Stability Effects of Mutations Designed to Increase thePrimary Sequence Symmetry Within the Core Region of a β-trefoil, 10Protein Sci. 2587-2599 (2001); Jaewon Kim et al., Alternative Type I andI′ Turn Conformations in the β8/β9 β-hairpin of Human Acidic FibroblastGrowth Factor, 11 Protein Sci. 459-466 (2002); Jaewon Kim et al.,Sequence swapping Does Not Result in Conformation Swapping for the β4/β5and β8/β9 β-hairpin Turns in Human Acidic Fibroblast Growth Factor, 14Protein Sci. 351-359 (2005). As a non-limiting example, replacement ofan amino acid comprising either the β4/β5 hairpin turn or β8/β9β-hairpin turn with a glycine results in increased stabilization of theβ-trefoil domain. Therefore, replacement of amino acids located in theβ4/β5 and β8/β9 β-hairpin turns of the β-trefoil domains present in thebinding domain of Clostridial toxins will increase binding activity ofsuch a modified Clostridial toxin by increasing the structural stabilityof the β-trefoil domain. The amino acid sequences comprising theβ-trefoil domains found in various Clostridial toxins are shown in Table2.

As is typical for proteins containing a β-trefoil fold, the overallamino acid sequence identity of the H_(CC) domain between Clostridialtoxins is low. However, key residues essential for binding activity havebeen identified by structural analysis and mutagenesis experiments, see,e.g., Krzysztof Ginalski et al., Structure-based Sequence Alignment forthe Beta-Trefoil Subdomain of the Clostridial Neurotoxin Family ProvidesResidue Level Information About the Putative Ganglioside Binding Site,482(1-2) FEBS Lett. 119-124 (2000). For example, analysis of the H_(CC)domain structure by crystallography identified five highly conservedresidues critical for forming a shallow surface pocket of a carbohydratebinding moiety. These polar residues make hydrogen bonds with thecarbohydrate ring. In BoNT/A these five polar residues are Glu 1203, Phe1252, Ser 1264, Tyr 1267 and Gly 1279, while in TeNT, these residues areAsp 1222, Thr 1270, Ser 1287, Tyr 1290 and Gly 1300. Additionally,tyrosine residues forming the hydrophilic wall of this pocket were alsoimportant (Trp 1266 of BoNT/A and Trp 1289 of TeNT) and tryptophanfluorescence quenching experiments indicated that Trp 1266 of BoNT/Abound carbohydrate molecules. In another studies, photoaffinity labelingexperiments revealed that Gln 1270 of BoNT/A and His 1293 of TeNT werealso involved in binding carbohydrate molecules. Mutagenesis experimentsdesigned to assay loss-of-function binding activity mutations confirmedthe importance of many of the residues described above for BoNT/A andTeNT and extended this analysis to BoNT/B (Glu 1190, His 1241, Typ 1262,Tyr 1263), see, e.g., Andreas Rummel et al., The H _(CC)-Domain ofBotulinum Neurotoxins A and B Exhibits a Singular Ganglioside BindingSite Displaying Serotype Specific Carbohydrate Interaction, 51(3) Mol.Microbiol. 631-643 (2004).

As used herein, the term “Clostridial toxin binding domain” means anyClostridial toxin polypeptide that can execute the cell binding step ofthe intoxication process, including, e.g., the selective binding of theClostridial toxin to a toxin-specific receptor located on the plasmamembrane surface of a target cell. Non-limiting examples of aClostridial toxin binding domain include, e.g., a Clostridial toxinH_(C) binding domain, such as, e.g., a BoNT/A H_(C) binding domain, aBoNT/B H_(C) binding domain, a BoNT/C1 H_(C) binding domain, a BoNT/DH_(C) binding domain, a BoNT/E H_(C) binding domain, a BoNT/F H_(C)binding domain, a BoNT/G H_(C) binding domain, a TeNT H_(C) bindingdomain, a BaNT H_(C) binding domain and a BuNT H_(C) binding domain.Other non-limiting examples of a Clostridial toxin H_(CC) bindingdomain, include a BoNT/A H_(CC) binding domain, a BoNT/B H_(CC) bindingdomain, a modified BoNT/C1 H_(CC) binding domain, a BoNT/D H_(CC)binding domain, a BoNT/E H_(CC) binding domain, a BoNT/F H_(CC) bindingdomain, a BoNT/G H_(CC) binding domain, a TeNT H_(CC) binding domain, aBaNT H_(CC) binding domain and a BuNT H_(CC) binding domain.

A Clostridial toxin binding domain includes, without limitation,naturally occurring Clostridial toxin binding domain variants, such as,e.g., Clostridial toxin binding domain isoforms and Clostridial toxinbinding domain subtypes; non-naturally occurring Clostridial toxinbinding domain variants, such as, e.g., conservative Clostridial toxinbinding domain variants, non-conservative Clostridial toxin bindingdomain variants, Clostridial toxin binding domain chimerics, activeClostridial toxin binding domain fragments thereof, or any combinationthereof.

As used herein, the term “Clostridial toxin binding domain variant,”whether naturally-occurring or non-naturally-occurring, means aClostridial toxin binding domain that has at least one amino acid changefrom the corresponding region of the disclosed reference sequences (seeTable 1) and can be described in percent identity to the correspondingregion of that reference sequence. Unless expressly indicated, allClostridial toxin binding domain variants disclosed in the presentspecification are capable of executing the cell binding step of theintoxication process, including, e.g., the selective binding of theClostridial toxin to a toxin-specific receptor located on the plasmamembrane surface of a target cell.

As a non-limiting example, a BoNT/A H_(C) binding domain variantcomprising amino acids 874-1296 of SEQ ID NO: 1 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 874-1296 ofSEQ ID NO: 1; a BoNT/B H_(C) binding domain variant comprising aminoacids 861-1291 of SEQ ID NO: 2 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 861-1291 of SEQ ID NO: 2;a BoNT/C1 H_(C) binding domain variant comprising amino acids 869-1291of SEQ ID NO: 3 will have at least one amino acid difference, such as,e.g., an amino acid substitution, deletion or addition, as compared tothe amino acid region 869-1291 of SEQ ID NO: 3; a BoNT/D H_(C) bindingdomain variant comprising amino acids 865-1276 of SEQ ID NO: 4 will haveat least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region865-1276 of SEQ ID NO: 4; a BoNT/E H_(C) binding domain variantcomprising amino acids 848-1252 of SEQ ID NO: 5 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 848-1252 ofSEQ ID NO: 5; a BoNT/F H_(C) binding domain variant comprising aminoacids 867-1274 of SEQ ID NO: 6 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 867-1274 of SEQ ID NO: 6;a BoNT/G H_(C) binding domain variant comprising amino acids 866-1297 ofSEQ ID NO: 7 will have at least one amino acid difference, such as,e.g., an amino acid substitution, deletion or addition, as compared tothe amino acid region 866-1297 of SEQ ID NO: 7; a TeNT H_(C) bindingdomain variant comprising amino acids 882-1315 of SEQ ID NO: 8 will haveat least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region882-1315 of SEQ ID NO: 8, a BaNT H_(C) binding domain variant comprisingamino acids 858-1269 of SEQ ID NO: 9 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 858-1268 of SEQ ID NO: 9,and a BuNT H_(C) binding domain variant comprising amino acids 848-1251of SEQ ID NO: 10 will have at least one amino acid difference, such as,e.g., an amino acid substitution, deletion or addition, as compared tothe amino acid region 848-1251 of SEQ ID NO: 10.

As another non-limiting example, a BoNT/A H_(CC) binding domaincomprising amino acids 1092-1296 of SEQ ID NO: 1 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 1092-1296 ofSEQ ID NO: 1; a modified BoNT/B H_(CC) binding domain comprising aminoacids 1079-1291 of SEQ ID NO: 2 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 1079-1291 of SEQ ID NO:2; a modified BoNT/C1 H_(CC) binding domain comprising amino acids1093-1291 of SEQ ID NO: 3 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 1093-1291 of SEQ ID NO: 3; a modifiedBoNT/D H_(CC) binding domain comprising amino acids 1080-1276 of SEQ IDNO: 4 will have at least one amino acid difference, such as, e.g., anamino acid substitution, deletion or addition, as compared to the aminoacid region 1080-1276 of SEQ ID NO: 4; a modified BoNT/E H_(CC) bindingdomain comprising amino acids 1067-1252 of SEQ ID NO: 5 will have atleast one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region1067-1252 of SEQ ID NO: 5; a modified BoNT/F H_(CC) binding domaincomprising amino acids 1087-1274 of SEQ ID NO: 6 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 1087-1274 ofSEQ ID NO: 6; a modified BoNT/G H_(CC) binding domain comprising aminoacids 1087-1297 of SEQ ID NO: 7 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 1087-1297 of SEQ ID NO:7; a modified TeNT H_(CC) binding domain comprising amino acids1109-1315 of SEQ ID NO: 8 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 1109-1315 of SEQ ID NO: 8, a modifiedBaNT H_(CC) binding domain comprising amino acids 1095-1269 of SEQ IDNO: 9 will have at least one amino acid difference, such as, e.g., anamino acid substitution, deletion or addition, as compared to the aminoacid region 1095-1268 of SEQ ID NO: 9, and a modified BuNT H_(CC)binding domain comprising amino acids 1086-1251 of SEQ ID NO: 10 willhave at least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region1086-1251 of SEQ ID NO: 10.

It is recognized by those of skill in the art that within each serotypeof Clostridial toxin there can be naturally occurring Clostridial toxinbinding domain variants that differ somewhat in their amino acidsequence, and also in the nucleic acids encoding these proteins. Forexample, there are presently four BoNT/A subtypes, BoNT/A1, BoNT/A2,BoNT/A3 and BoNT/A4, with specific binding domain subtypes showingapproximately 87% amino acid identity when compared to another BoNT/Abinding domain subtype. As used herein, the term “naturally occurringClostridial toxin binding domain variant” means any Clostridial toxinbinding domain produced by a naturally-occurring process, including,without limitation, Clostridial toxin binding domain isoforms producedfrom alternatively-spliced transcripts, Clostridial toxin binding domainisoforms produced by spontaneous mutation and Clostridial toxin bindingdomain subtypes. A naturally occurring Clostridial toxin binding domainvariant can function in substantially the same manner as the referenceClostridial toxin binding domain on which the naturally occurringClostridial toxin binding domain variant is based, and can besubstituted for the reference Clostridial toxin binding domain in anyaspect of the present invention. A naturally occurring Clostridial toxinbinding domain variant may substitute one or more amino acids, two ormore amino acids, three or more amino acids, four or more amino acids,five or more amino acids, ten or more amino acids, 20 or more aminoacids, 30 or more amino acids, 40 or more amino acids, 50 or more aminoacids or 100 or more amino acids from the reference Clostridial toxinbinding domain on which the naturally occurring Clostridial toxinbinding domain variant is based. A naturally occurring Clostridial toxinbinding domain variant can also substitute at least 10 contiguous aminoacids, at least 15 contiguous amino acids, at least 20 contiguous aminoacids, or at least 25 contiguous amino acids from the referenceClostridial toxin binding domain on which the naturally occurringClostridial toxin binding domain variant is based, that possess at least50% amino acid identity, 65% amino acid identity, 75% amino acididentity, 85% amino acid identity or 95% amino acid identity to thereference Clostridial toxin binding domain on which the naturallyoccurring Clostridial toxin binding domain variant is based.

A non-limiting examples of a naturally occurring Clostridial toxinbinding domain variant is a Clostridial toxin binding domain isoformsuch as, e.g., a BoNT/A binding domain isoform, a BoNT/B binding domainisoform, a BoNT/C1 binding domain isoform, a BoNT/D binding domainisoform, a BoNT/E binding domain isoform, a BoNT/F binding domainisoform, a BoNT/G binding domain isoform, a TeNT binding domain isoform,a BaNT binding domain isoform, and a BuNT binding domain isoform. AClostridial toxin binding domain isoform can function in substantiallythe same manner as the reference Clostridial toxin binding domain onwhich the Clostridial toxin binding domain isoform is based, and can besubstituted for the reference Clostridial toxin binding domain in anyaspect of the present invention.

Another non-limiting examples of a naturally occurring Clostridial toxinbinding domain variant is a Clostridial toxin binding domain subtypesuch as, e.g., a binding domain from subtype BoNT/A1, BoNT/A2, BoNT/A3and BoNT/A4; a binding domain from subtype BoNT/B1, BoNT/B2, BoNT/Bbivalent and BoNT/B nonproteolytic; a binding domain from subtypeBoNT/C1-1 and BoNT/C1-2; a binding domain from subtype BoNT/E1, BoNT/E2and BoNT/E3; and a binding domain from subtype BoNT/F1, BoNT/F2, BoNT/F3and BoNT/F4. A Clostridial toxin binding domain subtype can function insubstantially the same manner as the reference Clostridial toxin bindingdomain on which the Clostridial toxin binding domain subtype is based,and can be substituted for the reference Clostridial toxin bindingdomain in any aspect of the present invention.

As used herein, the term “non-naturally occurring Clostridial toxinbinding domain variant” means any Clostridial toxin binding domainproduced with the aid of human manipulation, including, withoutlimitation, Clostridial toxin binding domains produced by geneticengineering using random mutagenesis or rational design and Clostridialtoxin binding domains produced by chemical synthesis. Non-limitingexamples of non-naturally occurring Clostridial toxin binding domainvariants include, e.g., conservative Clostridial toxin binding domainvariants, non-conservative Clostridial toxin binding domain variants,Clostridial toxin binding domain chimeric variants and activeClostridial toxin binding domain fragments.

As used herein, the term “conservative Clostridial toxin binding domainvariant” means a Clostridial toxin binding domain that has at least oneamino acid substituted by another amino acid or an amino acid analogthat has at least one property similar to that of the original aminoacid from the reference Clostridial toxin binding domain sequence (Table1). Examples of properties include, without limitation, similar size,topography, charge, hydrophobicity, hydrophilicity, lipophilicity,covalent-bonding capacity, hydrogen-bonding capacity, a physicochemicalproperty, of the like, or any combination thereof. A conservativeClostridial toxin binding domain variant can function in substantiallythe same manner as the reference Clostridial toxin binding domain onwhich the conservative Clostridial toxin binding domain variant isbased, and can be substituted for the reference Clostridial toxinbinding domain in any aspect of the present invention. A conservativeClostridial toxin binding domain variant may substitute one or moreamino acids, two or more amino acids, three or more amino acids, four ormore amino acids, five or more amino acids, ten or more amino acids, 20or more amino acids, 30 or more amino acids, 40 or more amino acids, 50or more amino acids, 100 or more amino acids, 200 or more amino acids,300 or more amino acids, 400 or more amino acids, or 500 or more aminoacids from the reference Clostridial toxin binding domain on which theconservative Clostridial toxin binding domain variant is based. Aconservative Clostridial toxin binding domain variant can alsosubstitute at least 10 contiguous amino acids, at least 15 contiguousamino acids, at least 20 contiguous amino acids, or at least 25contiguous amino acids from the reference Clostridial toxin bindingdomain on which the conservative Clostridial toxin binding domainvariant is based, that possess at least 50% amino acid identity, 65%amino acid identity, 75% amino acid identity, 85% amino acid identity or95% amino acid identity to the reference Clostridial toxin bindingdomain on which the conservative Clostridial toxin binding domainvariant is based. Non-limiting examples of a conservative Clostridialtoxin binding domain variant include, e.g., conservative BoNT/A bindingdomain variants, conservative BoNT/B binding domain variants,conservative BoNT/C1 binding domain variants, conservative BoNT/Dbinding domain variants, conservative BoNT/E binding domain variants,conservative BoNT/F binding domain variants, conservative BoNT/G bindingdomain variants, conservative TeNT binding domain variants, conservativeBaNT binding domain variants and conservative BuNT binding domainvariants.

As used herein, the term “non-conservative Clostridial toxin bindingdomain variant” means a Clostridial toxin binding domain in which 1) atleast one amino acid is deleted from the reference Clostridial toxinbinding domain on which the non-conservative Clostridial toxin bindingdomain variant is based; 2) at least one amino acid added to thereference Clostridial toxin binding domain on which the non-conservativeClostridial toxin binding domain is based; or 3) at least one amino acidis substituted by another amino acid or an amino acid analog that doesnot share any property similar to that of the original amino acid fromthe reference Clostridial toxin binding domain sequence (Table 1). Anon-conservative Clostridial toxin binding domain variant can functionin substantially the same manner as the reference Clostridial toxinbinding domain on which the non-conservative Clostridial toxin bindingdomain variant is based, and can be substituted for the referenceClostridial toxin binding domain in any aspect of the present invention.A non-conservative Clostridial toxin binding domain variant can deleteone or more amino acids, two or more amino acids, three or more aminoacids, four or more amino acids, five or more amino acids, and ten ormore amino acids from the reference Clostridial toxin binding domain onwhich the non-conservative Clostridial toxin binding domain variant isbased. A non-conservative Clostridial toxin binding domain variant canadd one or more amino acids, two or more amino acids, three or moreamino acids, four or more amino acids, five or more amino acids, and tenor more amino acids to the reference Clostridial toxin binding domain onwhich the non-conservative Clostridial toxin binding domain variant isbased. A non-conservative Clostridial toxin binding domain variant maysubstitute one or more amino acids, two or more amino acids, three ormore amino acids, four or more amino acids, five or more amino acids,ten or more amino acids, 20 or more amino acids, 30 or more amino acids,40 or more amino acids, 50 or more amino acids, 100 or more amino acids,200 or more amino acids, 300 or more amino acids, 400 or more aminoacids, or 500 or more amino acids from the reference Clostridial toxinbinding domain on which the non-conservative Clostridial toxin bindingdomain variant is based. A non-conservative Clostridial toxin bindingdomain variant can also substitute at least 10 contiguous amino acids,at least 15 contiguous amino acids, at least 20 contiguous amino acids,or at least 25 contiguous amino acids from the reference Clostridialtoxin binding domain on which the non-conservative Clostridial toxinbinding domain variant is based, that possess at least 50% amino acididentity, 65% amino acid identity, 75% amino acid identity, 85% aminoacid identity or 95% amino acid identity to the reference Clostridialtoxin binding domain on which the non-conservative Clostridial toxinbinding domain variant is based. Non-limiting examples of anon-conservative Clostridial toxin binding domain variant include, e.g.,non-conservative BoNT/A binding domain variants, non-conservative BoNT/Bbinding domain variants, non-conservative BoNT/C1 binding domainvariants, non-conservative BoNT/D binding domain variants,non-conservative BoNT/E binding domain variants, non-conservative BoNT/Fbinding domain variants, non-conservative BoNT/G binding domainvariants, non-conservative TeNT binding domain variants,non-conservative BaNT binding domain variants and non-conservative BuNTbinding domain variants.

As used herein, the term “Clostridial toxin binding domain chimeric”means a polypeptide comprising at least a portion of a Clostridial toxinbinding domain and at least a portion of at least one other polypeptideto form a toxin binding domain with at least one property different fromthe reference Clostridial toxin binding domains of Table 1, with theproviso that this Clostridial toxin binding domain chimeric is stillcapable of executing the cell binding step of the intoxication process,including, e.g., the selective binding of the Clostridial toxin to atoxin-specific receptor located on the plasma membrane surface of atarget cell.

As used herein, the term “active Clostridial toxin binding domainfragment” means any of a variety of Clostridial toxin fragmentscomprising the binding domain can be useful in aspects of the presentinvention with the proviso that these active fragments can execute thecell binding step of the intoxication process, including, e.g., theselective binding of the Clostridial toxin to a toxin-specific receptorlocated on the plasma membrane surface of a target cell. For example,the H_(C) binding domains from the heavy chains of Clostridial toxinsare approximately 400-435 amino acids in length and comprise atranslocation domain (Table 1). Research has shown that the entirelength of a binding domain from a Clostridial toxin heavy chain is notnecessary for the translocating activity of the translocation domain.Thus, aspects of this embodiment can include Clostridial toxin bindingdomains comprising a translocation domain having a length of, e.g., atleast 350 amino acids, at least 375 amino acids, at least 400 aminoacids and at least 425 amino acids. Other aspects of this embodiment caninclude Clostridial toxin binding domains comprising translocationdomain having a length of, e.g., at most 350 amino acids, at most 375amino acids, at most 400 amino acids and at most 425 amino acids. Asanother example, the H_(CC) binding domain from the heavy chains ofClostridial toxins are approximately 165-195 amino acids in length andcomprise a binding domain (Table 1). Research has shown that the entirelength of a H_(CC) binding domain from a Clostridial toxin heavy chainis not necessary for the binding activity of the binding domain. Thus,aspects of this embodiment can include a Clostridial toxin H_(CC)binding domain comprising a binding domain having a length of, e.g., atleast 150 amino acids, at least 175 amino acids, at least 200 aminoacids and at least 225 amino acids. Other aspects of this embodiment caninclude a Clostridial toxin H_(CC) binding domain comprising a bindingdomain having a length of, e.g., at most 150 amino acids, at most 175amino acids, at most 200 amino acids and at most 225 amino acids.

Any of a variety of sequence alignment methods can be used to determinepercent identity of naturally-occurring Clostridial toxin binding domainvariants and non-naturally-occurring Clostridial toxin binding domainvariants, including, without limitation, global methods, local methodsand hybrid methods, such as, e.g., segment approach methods. Protocolsto determine percent identity are routine procedures within the scope ofone skilled in the art and from the teaching herein.

Thus, in an embodiment, a multivalent Clostridial toxin disclosed in thepresent specification comprises a Clostridial toxin binding domain. Inan aspect of this embodiment, a Clostridial toxin binding domaincomprises a naturally occurring Clostridial toxin binding domainvariant, such as, e.g., a Clostridial toxin binding domain isoform or aClostridial toxin binding domain subtype. In another aspect of thisembodiment, a Clostridial toxin binding domain comprises a non-naturallyoccurring Clostridial toxin binding domain variant, such as, e.g., aconservative Clostridial toxin binding domain variant, anon-conservative Clostridial toxin binding domain variant, a Clostridialtoxin chimeric binding domain, an active Clostridial toxin bindingdomain fragment, or any combination thereof.

In an embodiment, a binding domain comprises a BoNT/A binding domain. Inan aspect of this embodiment, a BoNT/A binding domain comprises a BoNT/AH_(C) binding domain. In aspects of this embodiment, a BoNT/A bindingdomain comprising BoNT/A H_(C) binding domain comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 874-1296of SEQ ID NO: 1, at least 75% amino acid identity with amino acids874-1296 of SEQ ID NO: 1, at least 80% amino acid identity with aminoacids 874-1296 of SEQ ID NO: 1, at least 85% amino acid identity withamino acids 874-1296 of SEQ ID NO: 1, at least 90% amino acid identitywith amino acids 874-1296 of SEQ ID NO: 1 or at least 95% amino acididentity with amino acids 874-1296 of SEQ ID NO: 1. In yet other aspectsof this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(C)binding domain comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 874-1296 of SEQ ID NO: 1, at most 75%amino acid identity with amino acids 874-1296 of SEQ ID NO: 1, at most80% amino acid identity with amino acids 874-1296 of SEQ ID NO: 1, atmost 85% amino acid identity with amino acids 874-1296 of SEQ ID NO: 1,at most 90% amino acid identity with amino acids 874-1296 of SEQ ID NO:1 or at most 95% amino acid identity with amino acids 874-1296 of SEQ IDNO: 1.

In other aspects of this embodiment, a BoNT/A binding domain comprisinga BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100, or 200 non-contiguous amino acid substitutions relative toamino acids 874-1296 of SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A binding domain comprising a BoNT/A H_(C) bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid substitutions relative to amino acids 874-1296of SEQ ID NO: 1. In yet other aspects of this embodiment, a BoNT/Abinding domain comprising a BoNT/A H_(C) binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 874-1296 of SEQ ID NO: 1. Inother aspects of this embodiment, a BoNT/A binding domain comprising aBoNT/A H_(C) binding domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 874-1296 of SEQ ID NO: 1. In still other aspects of thisembodiment, a BoNT/A binding domain comprising a BoNT/A H_(C) bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid additions relative to amino acids 874-1296 ofSEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A bindingdomain comprising a BoNT/A H_(C) binding domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 874-1296 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A binding domain comprisinga BoNT/A H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid substitutions relative to aminoacids 874-1296 of SEQ ID NO: 1. In other aspects of this embodiment, aBoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 874-1296 of SEQ ID NO: 1. In yetother aspects of this embodiment, a BoNT/A binding domain comprising aBoNT/A H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid deletions relative to aminoacids 874-1296 of SEQ ID NO: 1. In other aspects of this embodiment, aBoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 874-1296 of SEQ ID NO: 1. In stillother aspects of this embodiment, a BoNT/A binding domain comprising aBoNT/A H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid additions relative to aminoacids 874-1296 of SEQ ID NO: 1. In other aspects of this embodiment, aBoNT/A binding domain comprising a BoNT/A H_(C) binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 874-1296 of SEQ ID NO: 1.

In another aspect of this embodiment, a BoNT/A binding domain comprisesa BoNT/A H_(CC) binding domain. In aspects of this embodiment, a BoNT/Abinding domain comprising a BoNT/A H_(CC) binding domain comprises amodification of amino acids 1111-1296 of SEQ ID NO: 1. In another aspectof this embodiment, a BoNT/A binding domain comprising a BoNT/A H_(CC)binding domain comprises a α-fold motif of a β-trefoil domain of aBoNT/A H_(CC) binding domain, a β-fold motif of a β-trefoil domain of aBoNT/A H_(CC) binding domain, or a γ-fold motif of a β-trefoil domain ofa BoNT/A H_(CC) binding domain. In another aspect of this embodiment, aBoNT/A binding domain comprising a BoNT/A H_(CC) binding domaincomprises a modification to amino acids 1111-1162, amino acids1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1111-1162, amino acids1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1, at least 75% aminoacid identity with amino acids 1111-1162, amino acids 1179-1223, oramino acids 1237-1296 of SEQ ID NO: 1, at least 80% amino acid identitywith amino acids 1111-1162, amino acids 1179-1223, or amino acids1237-1296 of SEQ ID NO: 1, at least 85% amino acid identity with aminoacids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQID NO: 1, at least 90% amino acid identity with amino acids 1111-1162,amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1 or atleast 95% amino acid identity with amino acids 1111-1162, amino acids1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In yet otheraspects of this embodiment, a BoNT/A H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1111-1162, amino acids 1179-1223,or amino acids 1237-1296 of SEQ ID NO: 1, at most 75% amino acididentity with amino acids 1111-1162, amino acids 1179-1223, or aminoacids 1237-1296 of SEQ ID NO: 1, at most 80% amino acid identity withamino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296of SEQ ID NO: 1, at most 85% amino acid identity with amino acids1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO:1, at most 90% amino acid identity with amino acids 1111-1162, aminoacids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1 or at most 95%amino acid identity with amino acids 1111-1162, amino acids 1179-1223,or amino acids 1237-1296 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ IDNO: 1. In other aspects of this embodiment, a BoNT/A H_(CC) bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1111-1162, amino acids 1179-1223,or amino acids 1237-1296 of SEQ ID NO: 1. In yet other aspects of thisembodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 1111-1162, amino acids 1179-1223, oramino acids 1237-1296 of SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 1111-1162, amino acids 1179-1223, oramino acids 1237-1296 of SEQ ID NO: 1. In still other aspects of thisembodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 1111-1162, amino acids 1179-1223, oramino acids 1237-1296 of SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 1111-1162, amino acids 1179-1223, oramino acids 1237-1296 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1111-1162,amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. Inother aspects of this embodiment, a BoNT/A H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1111-1162,amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In yetother aspects of this embodiment, a BoNT/A H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1111-1162, aminoacids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1111-1162, aminoacids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In stillother aspects of this embodiment, a BoNT/A H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1111-1162, aminoacids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1111-1162, aminoacids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1.

In another embodiment, a BoNT/A binding domain comprising a BoNT/AH_(CC) binding domain comprises a β4/β5 hairpin turn of a β-trefoildomain of a BoNT/A H_(CC) binding domain or a β8/β9 hairpin turn of aβ-trefoil domain of a BoNT/A H_(CC) binding domain. In another aspect ofthis embodiment, a BoNT/A binding domain comprising a BoNT/A H_(CC)binding domain comprises a modification of amino acids 1163-1178 oramino acids 1224-1236 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1163-1178 or aminoacids 1224-1236 of SEQ ID NO: 1, at least 75% amino acid identity withamino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1, at least80% amino acid identity with amino acids 1163-1178 or amino acids1224-1236 of SEQ ID NO: 1, at least 85% amino acid identity with aminoacids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1, at least 90%amino acid identity with amino acids 1163-1178 or amino acids 1224-1236of SEQ ID NO: 1 or at least 95% amino acid identity with amino acids1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In yet other aspectsof this embodiment, a BoNT/A H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1163-1178 or amino acids 1224-1236of SEQ ID NO: 1, at most 75% amino acid identity with amino acids1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1, at most 80% aminoacid identity with amino acids 1163-1178 or amino acids 1224-1236 of SEQID NO: 1, at most 85% amino acid identity with amino acids 1163-1178 oramino acids 1224-1236 of SEQ ID NO: 1, at most 90% amino acid identitywith amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1 orat most 95% amino acid identity with amino acids 1163-1178 or aminoacids 1224-1236 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In other aspects ofthis embodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoilfold domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or 10 non-contiguous aminoacid substitutions relative to amino acids 1163-1178 or amino acids1224-1236 of SEQ ID NO: 1. In other aspects of this embodiment, anon-contiguous amino acid substitution of any amino acid from aminoacids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1 can be replacedwith glycine. In other aspects of this embodiment, a non-contiguousamino acid substitution of any hydrophobic amino acid from amino acids1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1 can be replaced withphenylalanine. In yet other aspects of this embodiment, a BoNT/A H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1163-1178 oramino acids 1224-1236 of SEQ ID NO: 1. In still other aspects of thisembodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidadditions relative to amino acids 1163-1178 or amino acids 1224-1236 ofSEQ ID NO: 1. In other aspects of this embodiment, a BoNT/A H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid additions relative toamino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid substitutions relative to amino acids 1163-1178 oramino acids 1224-1236 of SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to amino acids 1163-1178 or amino acids 1224-1236of SEQ ID NO: 1. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1163-1178 or aminoacids 1224-1236 of SEQ ID NO: 1 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1163-1178 or amino acids1224-1236 of SEQ ID NO: 1 can be replaced with phenylalanine. In yetother aspects of this embodiment, a BoNT/A H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid deletions relative to amino acids 1163-1178 oramino acids 1224-1236 of SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino aciddeletions relative to amino acids 1163-1178 or amino acids 1224-1236 ofSEQ ID NO: 1. In still other aspects of this embodiment, a BoNT/A H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid additions relative to amino acids 1163-1178 or amino acids1224-1236 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CC) binding domaincomprises a substitution of amino acid Trp 1101, Gly 1102, Leu 1105, Tyr1111, Tyr 1112, Gly 1158, Ile 1163, Asp 1179, Glu 1203, Phe 1252, Ser1264, Trp 1266, Tyr 1267, Gln 1270, Gly 1279 or Trp 1282, or anycombination thereof, the substitution enhancing the binding activity ofthe BoNT/A H_(CC) binding domain. In other aspects of this embodiment, aBoNT/A H_(CC) binding domain comprises a deletion of amino acid Trp1101, Gly 1102, Leu 1105, Tyr 1111, Tyr 1112, Gly 1158, Ile 1163, Asp1179, Glu 1203, Phe 1252, Ser 1264, Trp 1266, Tyr 1267, Gln 1270, Gly1279 or Trp 1282, or any combination thereof, the deletion enhancing thebinding activity of the BoNT/A H_(CC) binding domain.

In another embodiment, a binding domain comprises a BoNT/B bindingdomain. In an aspect of this embodiment, a BoNT/B binding domaincomprises a BoNT/B H_(C) binding domain. In other aspects of thisembodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) bindingdomain comprises a polypeptide, e.g., at least 70% amino acid identitywith amino acids 861-1291 of SEQ ID NO: 2, at least 75% amino acididentity with amino acids 861-1291 of SEQ ID NO: 2, at least 80% aminoacid identity with amino acids 861-1291 of SEQ ID NO: 2, at least 85%amino acid identity with amino acids 861-1291 of SEQ ID NO: 2, at least90% amino acid identity with amino acids 861-1291 of SEQ ID NO: 2 or atleast 95% amino acid identity with amino acids 861-1291 of SEQ ID NO: 2.In yet other aspects of this embodiment, a BoNT/B binding domaincomprising a BoNT/B H_(C) binding domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 861-1291 of SEQID NO: 2, at most 75% amino acid identity with amino acids 861-1291 ofSEQ ID NO: 2, at most 80% amino acid identity with amino acids 861-1291of SEQ ID NO: 2, at most 85% amino acid identity with amino acids861-1291 of SEQ ID NO: 2, at most 90% amino acid identity with aminoacids 861-1291 of SEQ ID NO: 2 or at most 95% amino acid identity withamino acids 861-1291 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B binding domain comprisinga BoNT/B H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100, or 200 non-contiguous amino acid substitutions relative toamino acids 861-1291 of SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid substitutions relative to amino acids 861-1291of SEQ ID NO: 2. In yet other aspects of this embodiment, a BoNT/Bbinding domain comprising a BoNT/B H_(C) binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 861-1291 of SEQ ID NO: 2. Inother aspects of this embodiment, a BoNT/B binding domain comprising aBoNT/B H_(C) binding domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 861-1291 of SEQ ID NO: 2. In still other aspects of thisembodiment, a BoNT/B binding domain comprising a BoNT/B H_(C) bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid additions relative to amino acids 861-1291 ofSEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B H_(C)binding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or200 non-contiguous amino acid additions relative to amino acids 861-1291of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B binding domain comprisinga BoNT/B H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid substitutions relative to aminoacids 861-1291 of SEQ ID NO: 2. In other aspects of this embodiment, aBoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 861-1291 of SEQ ID NO: 2. In yetother aspects of this embodiment, a BoNT/B binding domain comprising aBoNT/B H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid deletions relative to aminoacids 861-1291 of SEQ ID NO: 2. In other aspects of this embodiment, aBoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 861-1291 of SEQ ID NO: 2. In stillother aspects of this embodiment, a BoNT/B binding domain comprising aBoNT/B H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid additions relative to aminoacids 861-1291 of SEQ ID NO: 2. In other aspects of this embodiment, aBoNT/B binding domain comprising a BoNT/B H_(C) binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 861-1291 of SEQ ID NO: 2.

In another aspect of this embodiment, a BoNT/B binding domain comprisesa BoNT/B H_(CC) binding domain. In another aspect of this embodiment, aBoNT/B binding domain comprising a BoNT/B H_(CC) binding domaincomprises an α-fold motif of a β-trefoil domain of a BoNT/B H_(CC)binding domain, a β-fold motif of a β-trefoil domain of a BoNT/B H_(CC)binding domain, or a γ-fold motif of a β-trefoil domain of a BoNT/BH_(CC) binding domain. In another aspect of this embodiment, a BoNT/Bbinding domain comprising a BoNT/B H_(CC) binding domain comprises amodification to amino acids 1098-1147, amino acids 1166-1210, or aminoacids 1223-1291 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1098-1147, amino acids1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2, at least 75% aminoacid identity with amino acids 1098-1147, amino acids 1166-1210, oramino acids 1223-1291 of SEQ ID NO: 2, at least 80% amino acid identitywith amino acids 1098-1147, amino acids 1166-1210, or amino acids1223-1291 of SEQ ID NO: 2, at least 85% amino acid identity with aminoacids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQID NO: 2, at least 90% amino acid identity with amino acids 1098-1147,amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2 or atleast 95% amino acid identity with amino acids 1098-1147, amino acids1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In yet otheraspects of this embodiment, a BoNT/B H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1098-1147, amino acids 1166-1210,or amino acids 1223-1291 of SEQ ID NO: 2, at most 75% amino acididentity with amino acids 1098-1147, amino acids 1166-1210, or aminoacids 1223-1291 of SEQ ID NO: 2, at most 80% amino acid identity withamino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291of SEQ ID NO: 2, at most 85% amino acid identity with amino acids1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO:2, at most 90% amino acid identity with amino acids 1098-1147, aminoacids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2 or at most 95%amino acid identity with amino acids 1098-1147, amino acids 1166-1210,or amino acids 1223-1291 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO:2. In other aspects of this embodiment, a BoNT/B H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO:2. In yet other aspects of this embodiment, a BoNT/B H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQID NO: 2. In other aspects of this embodiment, a BoNT/B H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQID NO: 2. In still other aspects of this embodiment, a BoNT/B H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 1098-1147, amino acids 1166-1210, or amino acids1223-1291 of SEQ ID NO: 2. In other aspects of this embodiment, a BoNT/BH_(CC) binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 1098-1147, amino acids 1166-1210, or amino acids1223-1291 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1098-1147,amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. Inother aspects of this embodiment, a BoNT/B H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1098-1147,amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In yetother aspects of this embodiment, a BoNT/B H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1098-1147, aminoacids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1098-1147, aminoacids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In stillother aspects of this embodiment, a BoNT/B H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1098-1147, aminoacids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1098-1147, aminoacids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2.

In another embodiment, a binding domain comprising a BoNT/B H bindingdomain comprises a β4/β5 hairpin turn of a β-trefoil domain of a BoNT/BH_(CC) binding domain or a β8/β9 hairpin turn of a β-trefoil domain of aBoNT/B H_(CC) binding domain. In another aspect of this embodiment, abinding domain comprising a BoNT/B H_(CC) binding domain comprises amodification of amino acids 1148-1165 or amino acids 1211-1222 of SEQ IDNO: 2.

In other aspects of this embodiment, a BoNT/B H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1148-1165 or aminoacids 1211-1222 of SEQ ID NO: 2, at least 75% amino acid identity withamino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2, at least80% amino acid identity with amino acids 1148-1165 or amino acids1211-1222 of SEQ ID NO: 2, at least 85% amino acid identity with aminoacids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2, at least 90%amino acid identity with amino acids 1148-1165 or amino acids 1211-1222of SEQ ID NO: 2 or at least 95% amino acid identity with amino acids1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In yet other aspectsof this embodiment, a BoNT/B H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1148-1165 or amino acids 1211-1222of SEQ ID NO: 2, at most 75% amino acid identity with amino acids1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2, at most 80% aminoacid identity with amino acids 1148-1165 or amino acids 1211-1222 of SEQID NO: 2, at most 85% amino acid identity with amino acids 1148-1165 oramino acids 1211-1222 of SEQ ID NO: 2, at most 90% amino acid identitywith amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2 orat most 95% amino acid identity with amino acids 1148-1165 or aminoacids 1211-1222 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In other aspects ofthis embodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoilfold domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or 10 non-contiguous aminoacid substitutions relative to amino acids 1148-1165 or amino acids1211-1222 of SEQ ID NO: 2. In other aspects of this embodiment, anon-contiguous amino acid substitution of any amino acid from aminoacids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2 can be replacedwith glycine. In other aspects of this embodiment, a non-contiguousamino acid substitution of any hydrophobic amino acid from amino acids1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2 can be replaced withphenylalanine. In yet other aspects of this embodiment, a BoNT/B H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1148-1165 oramino acids 1211-1222 of SEQ ID NO: 2. In still other aspects of thisembodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidadditions relative to amino acids 1148-1165 or amino acids 1211-1222 ofSEQ ID NO: 2. In other aspects of this embodiment, a BoNT/B H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid additions relative toamino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid substitutions relative to amino acids 1148-1165 oramino acids 1211-1222 of SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to amino acids 1148-1165 or amino acids 1211-1222of SEQ ID NO: 2. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1148-1165 or aminoacids 1211-1222 of SEQ ID NO: 2 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1148-1165 or amino acids1211-1222 of SEQ ID NO: 2 can be replaced with phenylalanine. In yetother aspects of this embodiment, a BoNT/B H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid deletions relative to amino acids 1148-1165 oramino acids 1211-1222 of SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino aciddeletions relative to amino acids 1148-1165 or amino acids 1211-1222 ofSEQ ID NO: 2. In still other aspects of this embodiment, a BoNT/B H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid additions relative to amino acids 1148-1165 or amino acids1211-1222 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(CC) binding domaincomprises a substitution of amino acid Trp 1088, Gly 1089, Leu 1092, Tyr1098, Tyr 1099, Gly 1142, Ile 1147, Asp 1165, Glu 1191, Ile 1240, Ser1260, Trp 1262, Tyr 1263, Glu 1266, Gly 1277 or Trp 1280, or anycombination thereof, the substitution enhancing the binding capabilityof the BoNT/B H_(CC) binding domain. In other aspects of thisembodiment, a BoNT/B H_(CC) binding domain comprises a deletion of aminoacid Trp 1088, Gly 1089, Leu 1092, Tyr 1098, Tyr 1099, Gly 1142, Ile1147, Asp 1165, Glu 1191, Ile 1240, Ser 1260, Trp 1262, Tyr 1263, Glu1266, Gly 1277 or Trp 1280, or any combination thereof, the deletionenhancing the binding capability of the BoNT/B H_(CC) binding domain.

In another embodiment, a binding domain comprises a BoNT/C1 bindingdomain. In an aspect of this embodiment, a BoNT/C1 binding domaincomprises a BoNT/C1 H_(C) binding domain. In other aspects of thisembodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) bindingdomain comprises a polypeptide having, e.g., at least 70% amino acididentity with amino acids 869-1291 of SEQ ID NO: 3, at least 75% aminoacid identity with amino acids 869-1291 of SEQ ID NO: 3, at least 80%amino acid identity with amino acids 869-1291 of SEQ ID NO: 3, at least85% amino acid identity with amino acids 869-1291 of SEQ ID NO: 3, atleast 90% amino acid identity with amino acids 869-1291 of SEQ ID NO: 3or at least 95% amino acid identity with amino acids 869-1291 of SEQ IDNO: 3. In yet other aspects of this embodiment, a BoNT/C1 binding domaincomprising a BoNT/C1 H_(C) binding domain comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 869-1291of SEQ ID NO: 3, at most 75% amino acid identity with amino acids869-1291 of SEQ ID NO: 3, at most 80% amino acid identity with aminoacids 869-1291 of SEQ ID NO: 3, at most 85% amino acid identity withamino acids 869-1291 of SEQ ID NO: 3, at most 90% amino acid identitywith amino acids 869-1291 of SEQ ID NO: 3 or at most 95% amino acididentity with amino acids 869-1291 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 binding domain comprisinga BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100, or 200 non-contiguous amino acid substitutions relative toamino acids 869-1291 of SEQ ID NO: 3. In other aspects of thisembodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid substitutions relative to amino acids 869-1291of SEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1binding domain comprising a BoNT/C1 H_(C) binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 869-1291 of SEQ ID NO: 3. Inother aspects of this embodiment, a BoNT/C1 binding domain comprising aBoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 869-1291 of SEQ ID NO: 3. In still other aspects of thisembodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C) bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid additions relative to amino acids 869-1291 ofSEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 bindingdomain comprising a BoNT/C1 H_(C) binding domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 869-1291 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 binding domain comprisinga BoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid substitutions relative to aminoacids 869-1291 of SEQ ID NO: 3. In other aspects of this embodiment, aBoNT/C1 binding domain comprising a BoNT/C1 H_(C) binding domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid substitutions relative to amino acids 869-1291 of SEQ ID NO:3. In yet other aspects of this embodiment, a BoNT/C1 binding domaincomprising a BoNT/C1 H_(C) binding domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid deletionsrelative to amino acids 869-1291 of SEQ ID NO: 3. In other aspects ofthis embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(C)binding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or200 contiguous amino acid deletions relative to amino acids 869-1291 ofSEQ ID NO: 3. In still other aspects of this embodiment, a BoNT/C1binding domain comprising a BoNT/C1 H_(C) binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 869-1291 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 binding domain comprising aBoNT/C1 H_(C) binding domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid additions relative to aminoacids 869-1291 of SEQ ID NO: 3.

In another aspect of this embodiment, a BoNT/C1 binding domain comprisesa BoNT/C1 H_(CC) binding domain. In an aspect of this embodiment, aBoNT/C1 binding domain comprising a BoNT/C1 H_(CC) binding domaincomprises a modification of amino acids 1112-1291 of SEQ ID NO: 3. Inanother aspect of this embodiment, a BoNT/C1 binding domain comprising aBoNT/C1 H_(CC) binding domain comprises a α-fold motif of a β-trefoildomain of a BoNT/C1 H_(CC) binding domain, a β-fold motif of a β-trefoildomain of a BoNT/C1 H_(CC) binding domain, or a γ-fold motif of aβ-trefoil domain of a BoNT/C1 H_(CC) binding domain. In another aspectof this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(CC)binding domain comprises a modification to amino acids 1112-1150, aminoacids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1112-1150, amino acids1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3, at least 75% aminoacid identity with amino acids 1112-1150, amino acids 1167-1218, oramino acids 1230-1291 of SEQ ID NO: 3, at least 80% amino acid identitywith amino acids 1112-1150, amino acids 1167-1218, or amino acids1230-1291 of SEQ ID NO: 3, at least 85% amino acid identity with aminoacids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQID NO: 3, at least 90% amino acid identity with amino acids 1112-1150,amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3 or atleast 95% amino acid identity with amino acids 1112-1150, amino acids1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In yet otheraspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1112-1150, amino acids 1167-1218,or amino acids 1230-1291 of SEQ ID NO: 3, at most 75% amino acididentity with amino acids 1112-1150, amino acids 1167-1218, or aminoacids 1230-1291 of SEQ ID NO: 3, at most 80% amino acid identity withamino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291of SEQ ID NO: 3, at most 85% amino acid identity with amino acids1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO:3, at most 90% amino acid identity with amino acids 1112-1150, aminoacids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3 or at most 95%amino acid identity with amino acids 1112-1150, amino acids 1167-1218,or amino acids 1230-1291 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO:3. In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO:3. In yet other aspects of this embodiment, a BoNT/C1 H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQID NO: 3. In other aspects of this embodiment, a BoNT/C1 H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQID NO: 3. In still other aspects of this embodiment, a BoNT/C1 H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 1112-1150, amino acids 1167-1218, or amino acids1230-1291 of SEQ ID NO: 3. In other aspects of this embodiment, aBoNT/C1 H_(CC) binding domain comprising a β-trefoil fold domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 1112-1150, amino acids 1167-1218, oramino acids 1230-1291 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1112-1150,amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. Inother aspects of this embodiment, a BoNT/C1 H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1112-1150,amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In yetother aspects of this embodiment, a BoNT/C1 H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1112-1150, aminoacids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1112-1150, aminoacids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In stillother aspects of this embodiment, a BoNT/C1 H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1112-1150, aminoacids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1112-1150, aminoacids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3.

In another embodiment, a BoNT/C1 binding domain comprising a BoNT/C1H_(CC) binding domain comprises a β4/β5 hairpin turn of a β-trefoildomain of a BoNT/C1 H_(CC) binding domain or a β8/β9 hairpin turn of aβ-trefoil domain of a BoNT/C1 H_(CC) binding domain. In another aspectof this embodiment, a BoNT/C1 binding domain comprising a BoNT/C1 H_(CC)binding domain comprises a modification of amino acids 1151-1166 oramino acids 1219-1229 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1151-1166 or aminoacids 1219-1229 of SEQ ID NO: 3, at least 75% amino acid identity withamino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3, at least80% amino acid identity with amino acids 1151-1166 or amino acids1219-1229 of SEQ ID NO: 3, at least 85% amino acid identity with aminoacids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3, at least 90%amino acid identity with amino acids 1151-1166 or amino acids 1219-1229of SEQ ID NO: 3 or at least 95% amino acid identity with amino acids1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In yet other aspectsof this embodiment, a BoNT/C1 H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1151-1166 or amino acids 1219-1229of SEQ ID NO: 3, at most 75% amino acid identity with amino acids1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3, at most 80% aminoacid identity with amino acids 1151-1166 or amino acids 1219-1229 of SEQID NO: 3, at most 85% amino acid identity with amino acids 1151-1166 oramino acids 1219-1229 of SEQ ID NO: 3, at most 90% amino acid identitywith amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3 orat most 95% amino acid identity with amino acids 1151-1166 or aminoacids 1219-1229 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In other aspects ofthis embodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoilfold domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or 10 non-contiguous aminoacid substitutions relative to amino acids 1151-1166 or amino acids1219-1229 of SEQ ID NO: 3. In other aspects of this embodiment, anon-contiguous amino acid substitution of any amino acid from aminoacids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3 can be replacedwith glycine. In other aspects of this embodiment, a non-contiguousamino acid substitution of any hydrophobic amino acid from amino acids1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3 can be replaced withphenylalanine. In yet other aspects of this embodiment, a BoNT/C1 H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1151-1166 oramino acids 1219-1229 of SEQ ID NO: 3. In still other aspects of thisembodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidadditions relative to amino acids 1151-1166 or amino acids 1219-1229 ofSEQ ID NO: 3. In other aspects of this embodiment, a BoNT/C1 H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid additions relative toamino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid substitutions relative to amino acids 1151-1166 oramino acids 1219-1229 of SEQ ID NO: 3. In other aspects of thisembodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to amino acids 1151-1166 or amino acids 1219-1229of SEQ ID NO: 3. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1151-1166 or aminoacids 1219-1229 of SEQ ID NO: 3 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1151-1166 or amino acids1219-1229 of SEQ ID NO: 3 can be replaced with phenylalanine. In yetother aspects of this embodiment, a BoNT/C1 H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid deletions relative to amino acids 1151-1166 oramino acids 1219-1229 of SEQ ID NO: 3. In other aspects of thisembodiment, a BoNT/C1 H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino aciddeletions relative to amino acids 1151-1166 or amino acids 1219-1229 ofSEQ ID NO: 3. In still other aspects of this embodiment, a BoNT/C1H_(CC) binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid additions relative to amino acids 1151-1166 or amino acids1219-1229 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CC) binding domaincomprises a substitution of amino acid Trp 1102, Gly 1103, Leu 1106, Tyr1112, Tyr 1113, Gly 1145, Ile 1150, Asp 1166, Glu 1196, Ile 1247, Gly1256, Trp 1258, Tyr 1259, His 1261, Gly 1281 or Trp 1284, or anycombination thereof, the substitution enhancing the binding activity ofthe BoNT/C1 H_(CC) binding domain. In other aspects of this embodiment,a BoNT/C1 H_(CC) binding domain comprises a deletion of amino acid Trp1102, Gly 1103, Leu 1106, Tyr 1112, Tyr 1113, Gly 1145, Ile 1150, Asp1166, Glu 1196, Ile 1247, Gly 1256, Trp 1258, Tyr 1259, His 1261, Gly1281 or Trp 1284, or any combination thereof, the deletion enhancing thebinding activity of the BoNT/C1 H_(CC) binding domain.

In another embodiment, a binding domain comprises a BoNT/D bindingdomain. In an aspect of this embodiment, a BoNT/D binding domaincomprises a BoNT/D H_(C) binding domain. In other aspects of thisembodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) bindingdomain comprises a polypeptide having, e.g., at least 70% amino acididentity with amino acids 865-1276 of SEQ ID NO: 4, at least 75% aminoacid identity with amino acids 865-1276 of SEQ ID NO: 4, at least 80%amino acid identity with amino acids 865-1276 of SEQ ID NO: 4, at least85% amino acid identity with amino acids 865-1276 of SEQ ID NO: 4, atleast 90% amino acid identity with amino acids 865-1276 of SEQ ID NO: 4or at least 95% amino acid identity with amino acids 865-1276 of SEQ IDNO: 4. In yet other aspects of this embodiment, a BoNT/D binding domaincomprising a BoNT/D H_(C) binding domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 865-1276 of SEQID NO: 4, at most 75% amino acid identity with amino acids 865-1276 ofSEQ ID NO: 4, at most 80% amino acid identity with amino acids 865-1276of SEQ ID NO: 4, at most 85% amino acid identity with amino acids865-1276 of SEQ ID NO: 4, at most 90% amino acid identity with aminoacids 865-1276 of SEQ ID NO: 4 or at most 95% amino acid identity withamino acids 865-1276 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D binding domain comprisinga BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100, or 200 non-contiguous amino acid substitutions relative toamino acids 865-1276 of SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid substitutions relative to amino acids 865-1276of SEQ ID NO: 4. In yet other aspects of this embodiment, a BoNT/Dbinding domain comprising a BoNT/D H_(C) binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 865-1276 of SEQ ID NO: 4. Inother aspects of this embodiment, a BoNT/D binding domain comprising aBoNT/D H_(C) binding domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 865-1276 of SEQ ID NO: 4. In still other aspects of thisembodiment, a BoNT/D binding domain comprising a BoNT/D H_(C) bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid additions relative to amino acids 865-1276 ofSEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D bindingdomain comprising a BoNT/D H_(C) binding domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 865-1276 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D binding domain comprisinga BoNT/D H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid substitutions relative to aminoacids 865-1276 of SEQ ID NO: 4. In other aspects of this embodiment, aBoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 865-1276 of SEQ ID NO: 4. In yetother aspects of this embodiment, a BoNT/D binding domain comprising aBoNT/D H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid deletions relative to aminoacids 865-1276 of SEQ ID NO: 4. In other aspects of this embodiment, aBoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 865-1276 of SEQ ID NO: 4. In stillother aspects of this embodiment, a BoNT/D binding domain comprising aBoNT/D H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid additions relative to aminoacids 865-1276 of SEQ ID NO: 4. In other aspects of this embodiment, aBoNT/D binding domain comprising a BoNT/D H_(C) binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 865-1276 of SEQ ID NO: 4.

In another aspect of this embodiment, a BoNT/D binding domain comprisesa BoNT/D H_(CC) binding domain. In an aspect of this embodiment, aBoNT/D binding domain comprising a BoNT/D H_(CC) binding domaincomprises a modification of amino acids 1099-1276 of SEQ ID NO: 4. Inanother aspect of this embodiment, a BoNT/D binding domain comprising aBoNT/D H_(CC) binding domain comprises a α-fold motif of a β-trefoildomain of a BoNT/D H_(CC) binding domain, a β-fold motif of a β-trefoildomain of a BoNT/D H_(CC) binding domain, or a γ-fold motif of aβ-trefoil domain of a BoNT/D H_(CC) binding domain. In another aspect ofthis embodiment, a BoNT/D binding domain comprising a BoNT/D H_(CC)binding domain comprises a modification to amino acids 1099-1137, aminoacids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1099-1137, amino acids1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4, at least 75% aminoacid identity with amino acids 1099-1137, amino acids 1154-1207, oramino acids 1219-1276 of SEQ ID NO: 4, at least 80% amino acid identitywith amino acids 1099-1137, amino acids 1154-1207, or amino acids1219-1276 of SEQ ID NO: 4, at least 85% amino acid identity with aminoacids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQID NO: 4, at least 90% amino acid identity with amino acids 1099-1137,amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4 or atleast 95% amino acid identity with amino acids 1099-1137, amino acids1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In yet otheraspects of this embodiment, a BoNT/D H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1099-1137, amino acids 1154-1207,or amino acids 1219-1276 of SEQ ID NO: 4, at most 75% amino acididentity with amino acids 1099-1137, amino acids 1154-1207, or aminoacids 1219-1276 of SEQ ID NO: 4, at most 80% amino acid identity withamino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276of SEQ ID NO: 4, at most 85% amino acid identity with amino acids1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO:4, at most 90% amino acid identity with amino acids 1099-1137, aminoacids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4 or at most 95%amino acid identity with amino acids 1099-1137, amino acids 1154-1207,or amino acids 1219-1276 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO:4. In other aspects of this embodiment, a BoNT/D H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO:4. In yet other aspects of this embodiment, a BoNT/D H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQID NO: 4. In other aspects of this embodiment, a BoNT/D H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQID NO: 4. In still other aspects of this embodiment, a BoNT/D H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 1099-1137, amino acids 1154-1207, or amino acids1219-1276 of SEQ ID NO: 4. In other aspects of this embodiment, a BoNT/DH_(CC) binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 1099-1137, amino acids 1154-1207, or amino acids1219-1276 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1099-1137,amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. Inother aspects of this embodiment, a BoNT/D H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1099-1137,amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In yetother aspects of this embodiment, a BoNT/D H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1099-1137, aminoacids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1099-1137, aminoacids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In stillother aspects of this embodiment, a BoNT/D H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1099-1137, aminoacids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1099-1137, aminoacids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4.

In another embodiment, a BoNT/D binding domain comprising a BoNT/DH_(CC) binding domain comprises a β4/β5 hairpin turn of a β-trefoildomain of a BoNT/D H_(CC) binding domain or a β8/β9 hairpin turn of aβ-trefoil domain of a BoNT/D H_(CC) binding domain. In another aspect ofthis embodiment, a BoNT/D binding domain comprising a BoNT/D H_(CC)binding domain comprises a modification of amino acids 1138-1153 oramino acids 1208-1218 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1138-1153 or aminoacids 1208-1218 of SEQ ID NO: 4, at least 75% amino acid identity withamino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4, at least80% amino acid identity with amino acids 1138-1153 or amino acids1208-1218 of SEQ ID NO: 4, at least 85% amino acid identity with aminoacids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4, at least 90%amino acid identity with amino acids 1138-1153 or amino acids 1208-1218of SEQ ID NO: 4 or at least 95% amino acid identity with amino acids1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In yet other aspectsof this embodiment, a BoNT/D H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1138-1153 or amino acids 1208-1218of SEQ ID NO: 4, at most 75% amino acid identity with amino acids1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4, at most 80% aminoacid identity with amino acids 1138-1153 or amino acids 1208-1218 of SEQID NO: 4, at most 85% amino acid identity with amino acids 1138-1153 oramino acids 1208-1218 of SEQ ID NO: 4, at most 90% amino acid identitywith amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4 orat most 95% amino acid identity with amino acids 1138-1153 or aminoacids 1208-1218 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In other aspects ofthis embodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoilfold domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or 10 non-contiguous aminoacid substitutions relative to amino acids 1138-1153 or amino acids1208-1218 of SEQ ID NO: 4. In other aspects of this embodiment, anon-contiguous amino acid substitution of any amino acid from aminoacids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4 can be replacedwith glycine. In other aspects of this embodiment, a non-contiguousamino acid substitution of any hydrophobic amino acid from amino acids1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4 can be replaced withphenylalanine. In yet other aspects of this embodiment, a BoNT/D H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1138-1153 oramino acids 1208-1218 of SEQ ID NO: 4. In still other aspects of thisembodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidadditions relative to amino acids 1138-1153 or amino acids 1208-1218 ofSEQ ID NO: 4. In other aspects of this embodiment, a BoNT/D H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid additions relative toamino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid substitutions relative to amino acids 1138-1153 oramino acids 1208-1218 of SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to amino acids 1138-1153 or amino acids 1208-1218of SEQ ID NO: 4. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1138-1153 or aminoacids 1208-1218 of SEQ ID NO: 4 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1138-1153 or amino acids1208-1218 of SEQ ID NO: 4 can be replaced with phenylalanine. In yetother aspects of this embodiment, a BoNT/D H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid deletions relative to amino acids 1138-1153 oramino acids 1208-1218 of SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino aciddeletions relative to amino acids 1138-1153 or amino acids 1208-1218 ofSEQ ID NO: 4. In still other aspects of this embodiment, a BoNT/D H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid additions relative to amino acids 1138-1153 or amino acids1208-1218 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CC) binding domaincomprises a substitution of amino acid Trp 1089, Gly 1090, Leu 1093, Tyr1099, Tyr 1100, Gly 1132, Ile 1137, Asp 1153, Asn 1186, Lys 1236, Trp1238, Arg 1239, Phe 1242, Ser 1262 or Trp 1265, or any combinationthereof, the substitution enhancing the binding activity of the BoNT/DH_(CC) binding domain. In other aspects of this embodiment, a BoNT/DH_(CC) binding domain comprises a deletion of amino acid Trp 1089, Gly1090, Leu 1093, Tyr 1099, Tyr 1100, Gly 1132, Ile 1137, Asp 1153, Asn1186, Lys 1236, Trp 1238, Arg 1239, Phe 1242, Ser 1262 or Trp 1265, orany combination thereof, the deletion enhancing the binding activity ofthe BoNT/D H_(CC) binding domain.

In another embodiment, a binding domain comprises a BoNT/E bindingdomain. In an aspect of this embodiment, a BoNT/E binding domaincomprises a BoNT/E H_(C) binding domain. In other aspects of thisembodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprisesa polypeptide having, e.g., at least 70% amino acid identity with aminoacids 848-1252 of SEQ ID NO: 5, at least 75% amino acid identity withamino acids 848-1252 of SEQ ID NO: 5, at least 80% amino acid identitywith amino acids 848-1252 of SEQ ID NO: 5, at least 85% amino acididentity with amino acids 848-1252 of SEQ ID NO: 5, at least 90% aminoacid identity with amino acids 848-1252 of SEQ ID NO: 5 or at least 95%amino acid identity with amino acids 848-1252 of SEQ ID NO: 5. In yetother aspects of this embodiment, a BoNT/E binding domain comprising aBoNT/E H_(C) comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 848-1252 of SEQ ID NO: 5, at most 75%amino acid identity with amino acids 848-1252 of SEQ ID NO: 5, at most80% amino acid identity with amino acids 848-1252 of SEQ ID NO: 5, atmost 85% amino acid identity with amino acids 848-1252 of SEQ ID NO: 5,at most 90% amino acid identity with amino acids 848-1252 of SEQ ID NO:5 or at most 95% amino acid identity with amino acids 848-1252 of SEQ IDNO: 5.

In other aspects of this embodiment, a BoNT/E binding domain comprisinga BoNT/E H_(C) comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, or200 non-contiguous amino acid substitutions relative to amino acids848-1252 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/Ebinding domain comprising a BoNT/E H_(C) comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid substitutionsrelative to amino acids 848-1252 of SEQ ID NO: 5. In yet other aspectsof this embodiment, a BoNT/E binding domain comprising a BoNT/E H_(C)comprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid deletions relative to amino acids 848-1252 ofSEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E bindingdomain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50, 100 or 200 non-contiguous amino acid deletions relative toamino acids 848-1252 of SEQ ID NO: 5. In still other aspects of thisembodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 848-1252 of SEQ ID NO: 5. Inother aspects of this embodiment, a BoNT/E binding domain comprising aBoNT/E H_(C) comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or200 non-contiguous amino acid additions relative to amino acids 848-1252of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E binding domain comprisinga BoNT/E H_(C) comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or200 contiguous amino acid substitutions relative to amino acids 848-1252of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E bindingdomain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50, 100 or 200 contiguous amino acid substitutions relative toamino acids 848-1252 of SEQ ID NO: 5. In yet other aspects of thisembodiment, a BoNT/E binding domain comprising a BoNT/E H_(C) comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 848-1252 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E binding domain comprising a BoNT/EH_(C) comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid deletions relative to amino acids 848-1252 of SEQID NO: 5. In still other aspects of this embodiment, a BoNT/E bindingdomain comprising a BoNT/E H_(C) comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50, 100 or 200 contiguous amino acid additions relative to aminoacids 848-1252 of SEQ ID NO: 5. In other aspects of this embodiment, aBoNT/E binding domain comprising a BoNT/E H_(C) comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid additionsrelative to amino acids 848-1252 of SEQ ID NO: 5.

In another aspect of this embodiment, a BoNT/E binding domain comprisesa BoNT/E H_(CC) binding domain. In an aspect of this embodiment, aBoNT/E binding domain comprising a BoNT/E H_(CC) binding domaincomprises a modification of amino acids 1086-1252 of SEQ ID NO: 5. Inanother aspect of this embodiment, a BoNT/E binding domain comprising aBoNT/E H_(CC) binding domain comprises a α-fold motif of a β-trefoildomain of a BoNT/E H_(CC) binding domain, a β-fold motif of a β-trefoildomain of a BoNT/E H_(CC) binding domain, or a γ-fold motif of aβ-trefoil domain of a BoNT/E H_(CC) binding domain. In another aspect ofthis embodiment, a BoNT/E binding domain comprising a BoNT/E H_(CC)binding domain comprises a modification to amino acids 1086-1129, aminoacids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1086-1129, amino acids1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5, at least 75% aminoacid identity with amino acids 1086-1129, amino acids 1147-1190, oramino acids 1199-1252 of SEQ ID NO: 5, at least 80% amino acid identitywith amino acids 1086-1129, amino acids 1147-1190, or amino acids1199-1252 of SEQ ID NO: 5, at least 85% amino acid identity with aminoacids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQID NO: 5, at least 90% amino acid identity with amino acids 1086-1129,amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5 or atleast 95% amino acid identity with amino acids 1086-1129, amino acids1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In yet otheraspects of this embodiment, a BoNT/E H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1086-1129, amino acids 1147-1190,or amino acids 1199-1252 of SEQ ID NO: 5, at most 75% amino acididentity with amino acids 1086-1129, amino acids 1147-1190, or aminoacids 1199-1252 of SEQ ID NO: 5, at most 80% amino acid identity withamino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252of SEQ ID NO: 5, at most 85% amino acid identity with amino acids1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO:5, at most 90% amino acid identity with amino acids 1086-1129, aminoacids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5 or at most 95%amino acid identity with amino acids 1086-1129, amino acids 1147-1190,or amino acids 1199-1252 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO:5. In other aspects of this embodiment, a BoNT/E H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO:5. In yet other aspects of this embodiment, a BoNT/E H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQID NO: 5. In other aspects of this embodiment, a BoNT/E H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQID NO: 5. In still other aspects of this embodiment, a BoNT/E H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 1086-1129, amino acids 1147-1190, or amino acids1199-1252 of SEQ ID NO: 5. In other aspects of this embodiment, a BoNT/EH_(CC) binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 1086-1129, amino acids 1147-1190, or amino acids1199-1252 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1086-1129,amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. Inother aspects of this embodiment, a BoNT/E H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1086-1129,amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In yetother aspects of this embodiment, a BoNT/E H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1086-1129, aminoacids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1086-1129, aminoacids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In stillother aspects of this embodiment, a BoNT/E H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1086-1129, aminoacids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1086-1129, aminoacids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5.

In another embodiment, a BoNT/E binding domain comprising a BoNT/EH_(CC) binding domain comprises a β4/β5 hairpin turn of a β-trefoildomain of a BoNT/E H_(CC) binding domain or a β8/β9 hairpin turn of aβ-trefoil domain of a BoNT/E H_(CC) binding domain. In another aspect ofthis embodiment, a BoNT/E binding domain comprising a BoNT/E H_(CC)binding domain comprises a modification of amino acids 1130-1146 oramino acids 1191-1198 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1130-1146 or aminoacids 1191-1198 of SEQ ID NO: 5, at least 75% amino acid identity withamino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5, at least80% amino acid identity with amino acids 1130-1146 or amino acids1191-1198 of SEQ ID NO: 5, at least 85% amino acid identity with aminoacids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5, at least 90%amino acid identity with amino acids 1130-1146 or amino acids 1191-1198of SEQ ID NO: 5 or at least 95% amino acid identity with amino acids1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In yet other aspectsof this embodiment, a BoNT/E H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1130-1146 or amino acids 1191-1198of SEQ ID NO: 5, at most 75% amino acid identity with amino acids1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5, at most 80% aminoacid identity with amino acids 1130-1146 or amino acids 1191-1198 of SEQID NO: 5, at most 85% amino acid identity with amino acids 1130-1146 oramino acids 1191-1198 of SEQ ID NO: 5, at most 90% amino acid identitywith amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5 orat most 95% amino acid identity with amino acids 1130-1146 or aminoacids 1191-1198 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In other aspects ofthis embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoilfold domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or 10 non-contiguous aminoacid substitutions relative to amino acids 1130-1146 or amino acids1191-1198 of SEQ ID NO: 5. In other aspects of this embodiment, anon-contiguous amino acid substitution of any amino acid from aminoacids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5 can be replacedwith glycine. In other aspects of this embodiment, a non-contiguousamino acid substitution of any hydrophobic amino acid from amino acids1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5 can be replaced withphenylalanine. In yet other aspects of this embodiment, a BoNT/E H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1130-1146 oramino acids 1191-1198 of SEQ ID NO: 5. In still other aspects of thisembodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidadditions relative to amino acids 1130-1146 or amino acids 1191-1198 ofSEQ ID NO: 5. In other aspects of this embodiment, a BoNT/E H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid additions relative toamino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid substitutions relative to amino acids 1130-1146 oramino acids 1191-1198 of SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to amino acids 1130-1146 or amino acids 1191-1198of SEQ ID NO: 5. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1130-1146 or aminoacids 1191-1198 of SEQ ID NO: 5 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1130-1146 or amino acids1191-1198 of SEQ ID NO: 5 can be replaced with phenylalanine. In yetother aspects of this embodiment, a BoNT/E H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid deletions relative to amino acids 1130-1146 oramino acids 1191-1198 of SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E H binding domain comprising a β-trefoil fold domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid deletionsrelative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO:5. In still other aspects of this embodiment, a BoNT/E H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine or 10 contiguous amino acid additions relative to amino acids1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In other aspects ofthis embodiment, a BoNT/E H_(CC) binding domain comprising a β-trefoilfold domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or 10 contiguous amino acidadditions relative to amino acids 1130-1146 or amino acids 1191-1198 ofSEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CC) binding domaincomprises a substitution of amino acid Trp 1076, Gly 1077, Leu 1080, Tyr1086, Tyr 1087, Gly 1124, Ile 1129, Asp 1146, Glu 1172, Phe 1213, Ser1221, Trp 1223, Tyr 1224, His 1227, Gly 1236 or Trp 1239, or anycombination thereof, the substitution enhancing the binding activity ofthe BoNT/E H_(CC) binding domain. In other aspects of this embodiment, aBoNT/E H_(CC) binding domain comprises a deletion of amino acid Trp1076, Gly 1077, Leu 1080, Tyr 1086, Tyr 1087, Gly 1124, Ile 1129, Asp1146, Glu 1172, Phe 1213, Ser 1221, Trp 1223, Tyr 1224, His 1227, Gly1236 or Trp 1239, or any combination thereof, the deletion enhancing thebinding activity of the BoNT/E H_(CC) binding domain.

In another embodiment, a binding domain comprises a BoNT/F bindingdomain. In an aspect of this embodiment, a BoNT/F binding domaincomprises a BoNT/F H_(C) binding domain. In other aspects of thisembodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) bindingdomain comprises a polypeptide having, e.g., at least 70% amino acididentity with amino acids 867-1274 of SEQ ID NO: 6, at least 75% aminoacid identity with amino acids 867-1274 of SEQ ID NO: 6, at least 80%amino acid identity with amino acids 867-1274 of SEQ ID NO: 6, at least85% amino acid identity with amino acids 867-1274 of SEQ ID NO: 6, atleast 90% amino acid identity with amino acids 867-1274 of SEQ ID NO: 6or at least 95% amino acid identity with amino acids 867-1274 of SEQ IDNO: 6. In yet other aspects of this embodiment, a BoNT/F binding domaincomprising a BoNT/F H_(C) binding domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 867-1274 of SEQID NO: 6, at most 75% amino acid identity with amino acids 867-1274 ofSEQ ID NO: 6, at most 80% amino acid identity with amino acids 867-1274of SEQ ID NO: 6, at most 85% amino acid identity with amino acids867-1274 of SEQ ID NO: 6, at most 90% amino acid identity with aminoacids 867-1274 of SEQ ID NO: 6 or at most 95% amino acid identity withamino acids 867-1274 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F binding domain comprisinga BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100, or 200 non-contiguous amino acid substitutions relative toamino acids 867-1274 of SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid substitutions relative to amino acids 867-1274of SEQ ID NO: 6. In yet other aspects of this embodiment, a BoNT/Fbinding domain comprising a BoNT/F H_(C) binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 867-1274 of SEQ ID NO: 6. Inother aspects of this embodiment, a BoNT/F binding domain comprising aBoNT/F H_(C) binding domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 867-1274 of SEQ ID NO: 6. In still other aspects of thisembodiment, a BoNT/F binding domain comprising a BoNT/F H_(C) bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid additions relative to amino acids 867-1274 ofSEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F bindingdomain comprising a BoNT/F H_(C) binding domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 867-1274 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F binding domain comprisinga BoNT/F H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid substitutions relative to aminoacids 867-1274 of SEQ ID NO: 6. In other aspects of this embodiment, aBoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 867-1274 of SEQ ID NO: 6. In yetother aspects of this embodiment, a BoNT/F binding domain comprising aBoNT/F H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid deletions relative to aminoacids 867-1274 of SEQ ID NO: 6. In other aspects of this embodiment, aBoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 867-1274 of SEQ ID NO: 6. In stillother aspects of this embodiment, a BoNT/F binding domain comprising aBoNT/F H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid additions relative to aminoacids 867-1274 of SEQ ID NO: 6. In other aspects of this embodiment, aBoNT/F binding domain comprising a BoNT/F H_(C) binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 867-1274 of SEQ ID NO: 6.

In another aspect of this embodiment, a BoNT/F binding domain comprisesa BoNT/F H_(CC) binding domain. In an aspect of this embodiment, aBoNT/F binding domain comprising a BoNT/F H_(CC) binding domaincomprises a modification of amino acids 1106-1274 of SEQ ID NO: 6. Inanother aspect of this embodiment, a BoNT/F binding domain comprising aBoNT/F H_(CC) binding domain comprises a α-fold motif of a β-trefoildomain of a BoNT/F H_(CC) binding domain, a β-fold motif of a β-trefoildomain of a BoNT/F H_(CC) binding domain, or a γ-fold motif of aβ-trefoil domain of a BoNT/F H_(CC) binding domain. In another aspect ofthis embodiment, a BoNT/F binding domain comprising a BoNT/F H_(CC)binding domain comprises a modification to amino acids 1106-1152, aminoacids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1106-1152, amino acids1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6, at least 75% aminoacid identity with amino acids 1106-1152, amino acids 1172-1213, oramino acids 1222-1274 of SEQ ID NO: 6, at least 80% amino acid identitywith amino acids 1106-1152, amino acids 1172-1213, or amino acids1222-1274 of SEQ ID NO: 6, at least 85% amino acid identity with aminoacids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQID NO: 6, at least 90% amino acid identity with amino acids 1106-1152,amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6 or atleast 95% amino acid identity with amino acids 1106-1152, amino acids1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In yet otheraspects of this embodiment, a BoNT/F H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1106-1152, amino acids 1172-1213,or amino acids 1222-1274 of SEQ ID NO: 6, at most 75% amino acididentity with amino acids 1106-1152, amino acids 1172-1213, or aminoacids 1222-1274 of SEQ ID NO: 6, at most 80% amino acid identity withamino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274of SEQ ID NO: 6, at most 85% amino acid identity with amino acids1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO:6, at most 90% amino acid identity with amino acids 1106-1152, aminoacids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6 or at most 95%amino acid identity with amino acids 1106-1152, amino acids 1172-1213,or amino acids 1222-1274 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO:6. In other aspects of this embodiment, a BoNT/F H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO:6. In yet other aspects of this embodiment, a BoNT/F H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQID NO: 6. In other aspects of this embodiment, a BoNT/F H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQID NO: 6. In still other aspects of this embodiment, a BoNT/F H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 1106-1152, amino acids 1172-1213, or amino acids1222-1274 of SEQ ID NO: 6. In other aspects of this embodiment, a BoNT/FH_(CC) binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 1106-1152, amino acids 1172-1213, or amino acids1222-1274 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1106-1152,amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. Inother aspects of this embodiment, a BoNT/F H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1106-1152,amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In yetother aspects of this embodiment, a BoNT/F H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1106-1152, aminoacids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1106-1152, aminoacids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In stillother aspects of this embodiment, a BoNT/F H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1106-1152, aminoacids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1106-1152, aminoacids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6.

In another embodiment, a BoNT/F binding domain comprises a BoNT/F H_(CC)binding domain of comprises a β4/β5 hairpin turn of a β-trefoil domainof a BoNT/F H_(CC) binding domain or a β8/β9 hairpin turn of a β-trefoildomain of a BoNT/F H_(CC) binding domain. In another aspect of thisembodiment, a BoNT/F binding domain comprising a BoNT/F H_(CC) bindingdomain comprises a modification of amino acids 1153-1171 or amino acids1214-1221 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1153-1171 or aminoacids 1214-1221 of SEQ ID NO: 6, at least 75% amino acid identity withamino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6, at least80% amino acid identity with amino acids 1153-1171 or amino acids1214-1221 of SEQ ID NO: 6, at least 85% amino acid identity with aminoacids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6, at least 90%amino acid identity with amino acids 1153-1171 or amino acids 1214-1221of SEQ ID NO: 6 or at least 95% amino acid identity with amino acids1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In yet other aspectsof this embodiment, a BoNT/F H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1153-1171 or amino acids 1214-1221of SEQ ID NO: 6, at most 75% amino acid identity with amino acids1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6, at most 80% aminoacid identity with amino acids 1153-1171 or amino acids 1214-1221 of SEQID NO: 6, at most 85% amino acid identity with amino acids 1153-1171 oramino acids 1214-1221 of SEQ ID NO: 6, at most 90% amino acid identitywith amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6 orat most 95% amino acid identity with amino acids 1153-1171 or aminoacids 1214-1221 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In other aspects ofthis embodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoilfold domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or 10 non-contiguous aminoacid substitutions relative to amino acids 1153-1171 or amino acids1214-1221 of SEQ ID NO: 6. In other aspects of this embodiment, anon-contiguous amino acid substitution of any amino acid from aminoacids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6 can be replacedwith glycine. In other aspects of this embodiment, a non-contiguousamino acid substitution of any hydrophobic amino acid from amino acids1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6 can be replaced withphenylalanine. In yet other aspects of this embodiment, a BoNT/F H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1153-1171 oramino acids 1214-1221 of SEQ ID NO: 6. In still other aspects of thisembodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidadditions relative to amino acids 1153-1171 or amino acids 1214-1221 ofSEQ ID NO: 6. In other aspects of this embodiment, a BoNT/F H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid additions relative toamino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid substitutions relative to amino acids 1153-1171 oramino acids 1214-1221 of SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to amino acids 1153-1171 or amino acids 1214-1221of SEQ ID NO: 6. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1153-1171 or aminoacids 1214-1221 of SEQ ID NO: 6 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1153-1171 or amino acids1214-1221 of SEQ ID NO: 6 can be replaced with phenylalanine. In yetother aspects of this embodiment, a BoNT/F H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid deletions relative to amino acids 1153-1171 oramino acids 1214-1221 of SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino aciddeletions relative to amino acids 1153-1171 or amino acids 1214-1221 ofSEQ ID NO: 6. In still other aspects of this embodiment, a BoNT/F H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid additions relative to amino acids 1153-1171 or amino acids1214-1221 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CC) binding domaincomprises a substitution of amino acid Trp 1096, Gly 1097, Leu 1100, Tyr1106, Tyr 1107, Gly 1147, Ile 1152, Asp 1171, Glu 1195, Phe 1237, Ser1245, Trp 1247, Tyr 1248, Asn 1251, Gly 1260 or Trp 1263, or anycombination thereof, the substitution enhancing the binding activity ofthe BoNT/F H_(CC) binding domain. In other aspects of this embodiment, aBoNT/F H_(CC) binding domain comprises a deletion of amino acid Trp1096, Gly 1097, Leu 1100, Tyr 1106, Tyr 1107, Gly 1147, Ile 1152, Asp1171, Glu 1195, Phe 1237, Ser 1245, Trp 1247, Tyr 1248, Asn 1251, Gly1260 or Trp 1263, or any combination thereof, the deletion enhancing thebinding activity of the BoNT/F H_(CC) binding domain.

In another embodiment, a binding domain comprises a BoNT/G bindingdomain. In an aspect of this embodiment, a BoNT/G binding domaincomprises a BoNT/G H_(C) binding domain. In other aspects of thisembodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) bindingdomain comprises a polypeptide having, e.g., at least 70% amino acididentity with amino acids 866-1297 of SEQ ID NO: 7, at least 75% aminoacid identity with amino acids 866-1297 of SEQ ID NO: 7, at least 80%amino acid identity with amino acids 866-1297 of SEQ ID NO: 7, at least85% amino acid identity with amino acids 866-1297 of SEQ ID NO: 7, atleast 90% amino acid identity with amino acids 866-1297 of SEQ ID NO: 7or at least 95% amino acid identity with amino acids 866-1297 of SEQ IDNO: 7. In yet other aspects of this embodiment, a BoNT/G binding domaincomprising a BoNT/G H_(C) binding domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 866-1297 of SEQID NO: 7, at most 75% amino acid identity with amino acids 866-1297 ofSEQ ID NO: 7, at most 80% amino acid identity with amino acids 866-1297of SEQ ID NO: 7, at most 85% amino acid identity with amino acids866-1297 of SEQ ID NO: 7, at most 90% amino acid identity with aminoacids 866-1297 of SEQ ID NO: 7 or at most 95% amino acid identity withamino acids 866-1297 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G binding domain comprisinga BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100, or 200 non-contiguous amino acid substitutions relative toamino acids 866-1297 of SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid substitutions relative to amino acids 866-1297of SEQ ID NO: 7. In yet other aspects of this embodiment, a BoNT/Gbinding domain comprising a BoNT/G H_(C) binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 866-1297 of SEQ ID NO: 7. Inother aspects of this embodiment, a BoNT/G binding domain comprising aBoNT/G H_(C) binding domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 866-1297 of SEQ ID NO: 7. In still other aspects of thisembodiment, a BoNT/G binding domain comprising a BoNT/G H_(C) bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid additions relative to amino acids 866-1297 ofSEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G bindingdomain comprising a BoNT/G H_(C) binding domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 866-1297 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G binding domain comprisinga BoNT/G H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid substitutions relative to aminoacids 866-1297 of SEQ ID NO: 7. In other aspects of this embodiment, aBoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 866-1297 of SEQ ID NO: 7. In yetother aspects of this embodiment, a BoNT/G binding domain comprising aBoNT/G H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid deletions relative to aminoacids 866-1297 of SEQ ID NO: 7. In other aspects of this embodiment, aBoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 866-1297 of SEQ ID NO: 7. In stillother aspects of this embodiment, a BoNT/G binding domain comprising aBoNT/G H_(C) binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid additions relative to aminoacids 866-1297 of SEQ ID NO: 7. In other aspects of this embodiment, aBoNT/G binding domain comprising a BoNT/G H_(C) binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 866-1297 of SEQ ID NO: 7.

In another aspect of this embodiment, a BoNT/G binding domain comprisesa BoNT/G H_(CC) binding domain. In an aspect of this embodiment, aBoNT/G binding domain comprising a BoNT/G H_(CC) binding domaincomprises a modification of amino acids 1106-1297 of SEQ ID NO: 7. Inanother aspect of this embodiment, a BoNT/G binding domain comprising aBoNT/G H_(CC) binding domain comprises a α-fold motif of a β-trefoildomain of a BoNT/G H_(CC) binding domain, a β-fold motif of a β-trefoildomain of a BoNT/G H_(CC) binding domain, or a γ-fold motif of aβ-trefoil domain of a BoNT/G H_(CC) binding domain. In another aspect ofthis embodiment, a BoNT/G binding domain comprising a BoNT/G H_(CC)binding domain comprises a modification to amino acids 1106-1153, aminoacids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1106-1153, amino acids1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7, at least 75% aminoacid identity with amino acids 1106-1153, amino acids 1173-1218, oramino acids 1231-1297 of SEQ ID NO: 7, at least 80% amino acid identitywith amino acids 1106-1153, amino acids 1173-1218, or amino acids1231-1297 of SEQ ID NO: 7, at least 85% amino acid identity with aminoacids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQID NO: 7, at least 90% amino acid identity with amino acids 1106-1153,amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7 or atleast 95% amino acid identity with amino acids 1106-1153, amino acids1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In yet otheraspects of this embodiment, a BoNT/G H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1106-1153, amino acids 1173-1218,or amino acids 1231-1297 of SEQ ID NO: 7, at most 75% amino acididentity with amino acids 1106-1153, amino acids 1173-1218, or aminoacids 1231-1297 of SEQ ID NO: 7, at most 80% amino acid identity withamino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297of SEQ ID NO: 7, at most 85% amino acid identity with amino acids1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO:7, at most 90% amino acid identity with amino acids 1106-1153, aminoacids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7 or at most 95%amino acid identity with amino acids 1106-1153, amino acids 1173-1218,or amino acids 1231-1297 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO:7. In other aspects of this embodiment, a BoNT/G H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO:7. In yet other aspects of this embodiment, a BoNT/G H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQID NO: 7. In other aspects of this embodiment, a BoNT/G H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQID NO: 7. In still other aspects of this embodiment, a BoNT/G H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 1106-1153, amino acids 1173-1218, or amino acids1231-1297 of SEQ ID NO: 7. In other aspects of this embodiment, a BoNT/GH_(CC) binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 1106-1153, amino acids 1173-1218, or amino acids1231-1297 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1106-1153,amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. Inother aspects of this embodiment, a BoNT/G H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1106-1153,amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In yetother aspects of this embodiment, a BoNT/G H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1106-1153, aminoacids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1106-1153, aminoacids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In stillother aspects of this embodiment, a BoNT/G H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1106-1153, aminoacids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1106-1153, aminoacids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7.

In another embodiment, a BoNT/G binding domain comprising a BoNT/GH_(CC) binding domain comprises a β4/β5 hairpin turn of a β-trefoildomain of a BoNT/G H_(CC) binding domain or a β8/β9 hairpin turn of aβ-trefoil domain of a BoNT/G H_(CC) binding domain. In another aspect ofthis embodiment, a BoNT/G binding domain comprising a BoNT/G H_(CC)binding domain comprises a modification of amino acids 1154-1172 oramino acids 1219-1230 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1154-1172 or aminoacids 1219-1230 of SEQ ID NO: 7, at least 75% amino acid identity withamino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7, at least80% amino acid identity with amino acids 1154-1172 or amino acids1219-1230 of SEQ ID NO: 7, at least 85% amino acid identity with aminoacids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7, at least 90%amino acid identity with amino acids 1154-1172 or amino acids 1219-1230of SEQ ID NO: 7 or at least 95% amino acid identity with amino acids1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In yet other aspectsof this embodiment, a BoNT/G H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1154-1172 or amino acids 1219-1230of SEQ ID NO: 7, at most 75% amino acid identity with amino acids1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7, at most 80% aminoacid identity with amino acids 1154-1172 or amino acids 1219-1230 of SEQID NO: 7, at most 85% amino acid identity with amino acids 1154-1172 oramino acids 1219-1230 of SEQ ID NO: 7, at most 90% amino acid identitywith amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7 orat most 95% amino acid identity with amino acids 1154-1172 or aminoacids 1219-1230 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In other aspects ofthis embodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoilfold domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or 10 non-contiguous aminoacid substitutions relative to amino acids 1154-1172 or amino acids1219-1230 of SEQ ID NO: 7. In other aspects of this embodiment, anon-contiguous amino acid substitution of any amino acid from aminoacids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7 can be replacedwith glycine. In other aspects of this embodiment, a non-contiguousamino acid substitution of any hydrophobic amino acid from amino acids1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7 can be replaced withphenylalanine. In yet other aspects of this embodiment, a BoNT/G H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1154-1172 oramino acids 1219-1230 of SEQ ID NO: 7. In still other aspects of thisembodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidadditions relative to amino acids 1154-1172 or amino acids 1219-1230 ofSEQ ID NO: 7. In other aspects of this embodiment, a BoNT/G H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid additions relative toamino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid substitutions relative to amino acids 1154-1172 oramino acids 1219-1230 of SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to amino acids 1154-1172 or amino acids 1219-1230of SEQ ID NO: 7. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1154-1172 or aminoacids 1219-1230 of SEQ ID NO: 7 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1154-1172 or amino acids1219-1230 of SEQ ID NO: 7 can be replaced with phenylalanine. In yetother aspects of this embodiment, a BoNT/G H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid deletions relative to amino acids 1154-1172 oramino acids 1219-1230 of SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino aciddeletions relative to amino acids 1154-1172 or amino acids 1219-1230 ofSEQ ID NO: 7. In still other aspects of this embodiment, a BoNT/G H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid additions relative to amino acids 1154-1172 or amino acids1219-1230 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CC) binding domaincomprises a substitution of amino acid Trp 1096, Gly 1097, Leu 1100, Tyr1106, Tyr 1107, Gly 1148, Ile 1153, Asp 1172, Gln 1198, Ile 1245, Ser1266, Trp 1268, Tyr 1269, Arg 1272, Gly 1283 or Trp 1285, or anycombination thereof, the substitution enhancing the binding activity ofthe BoNT/G H_(CC) binding domain. In other aspects of this embodiment, aBoNT/G H_(CC) binding domain comprises a deletion of amino acid Trp1096, Gly 1097, Leu 1100, Tyr 1106, Tyr 1107, Gly 1148, Ile 1153, Asp1172, Gln 1198, Ile 1245, Ser 1266, Trp 1268, Tyr 1269, Arg 1272, Gly1283 or Trp 1285, or any combination thereof, the deletion enhancing thebinding activity of the BoNT/G H_(CC) binding domain.

In another embodiment, a binding domain comprises a TeNT binding domain.In an aspect of this embodiment, a TeNT binding domain comprises a TeNTH_(C) binding domain. In other aspects of this embodiment, a TeNTbinding domain comprising a TeNT H_(C) binding domain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 882-1315 of SEQ ID NO: 8, at least 75% amino acid identity withamino acids 882-1315 of SEQ ID NO: 8, at least 80% amino acid identitywith amino acids 882-1315 of SEQ ID NO: 8, at least 85% amino acididentity with amino acids 882-1315 of SEQ ID NO: 8, at least 90% aminoacid identity with amino acids 882-1315 of SEQ ID NO: 8 or at least 95%amino acid identity with amino acids 882-1315 of SEQ ID NO: 8. In yetother aspects of this embodiment, a TeNT binding domain comprising aTeNT H_(C) binding domain comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 882-1315 of SEQ ID NO: 8, atmost 75% amino acid identity with amino acids 882-1315 of SEQ ID NO: 8,at most 80% amino acid identity with amino acids 882-1315 of SEQ ID NO:8, at most 85% amino acid identity with amino acids 882-1315 of SEQ IDNO: 8, at most 90% amino acid identity with amino acids 882-1315 of SEQID NO: 8 or at most 95% amino acid identity with amino acids 882-1315 ofSEQ ID NO: 8.

In other aspects of this embodiment, a TeNT binding domain comprising aTeNT H_(C) binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100, or 200 non-contiguous amino acid substitutions relative toamino acids 882-1315 of SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT binding domain comprising a TeNT H_(C) binding domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid substitutions relative to amino acids 882-1315of SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT bindingdomain comprising a TeNT H_(C) binding domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 882-1315 of SEQ ID NO: 8. In other aspects ofthis embodiment, a TeNT binding domain comprising a TeNT H_(C) bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid deletions relative to amino acids 882-1315 ofSEQ ID NO: 8. In still other aspects of this embodiment, a TeNT bindingdomain comprising a TeNT H_(C) binding domain comprises a polypeptide ehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 882-1315 of SEQ ID NO: 8. In other aspects ofthis embodiment, a TeNT binding domain comprising a TeNT H_(C) bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid additions relative to amino acids 882-1315 ofSEQ ID NO: 8.

In other aspects of this embodiment, a TeNT binding domain comprising aTeNT H_(C) binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 contiguous amino acid substitutions relative to aminoacids 882-1315 of SEQ ID NO: 8. In other aspects of this embodiment, aTeNT binding domain comprising a TeNT H_(C) binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 882-1315 of SEQ ID NO: 8. In yetother aspects of this embodiment, a TeNT binding domain comprising aTeNT H_(C) binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 contiguous amino acid deletions relative to amino acids882-1315 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNTbinding domain comprising a TeNT H_(C) binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 882-1315 of SEQ ID NO: 8. In stillother aspects of this embodiment, a TeNT binding domain comprising aTeNT H_(C) binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 contiguous amino acid additions relative to amino acids882-1315 of SEQ ID NO: 8. In other aspects of this embodiment, a TeNTbinding domain comprising a TeNT H_(C) binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 882-1315 of SEQ ID NO: 8.

In another aspect of this embodiment, a TeNT binding domain comprises aTeNT H_(CC) binding domain. In an aspect of this embodiment, a TeNTbinding domain comprising a TeNT H_(CC) binding domain comprises amodification of amino acids 1128-1315 of SEQ ID NO: 8. In another aspectof this embodiment, a TeNT H_(CC) binding domain comprises a α-foldmotif of a β-trefoil domain of a TeNT H_(CC) binding domain, a β-foldmotif of a β-trefoil domain of a TeNT H_(CC) binding domain, or a γ-foldmotif of a β-trefoil domain of a TeNT H_(CC) binding domain. In anotheraspect of this embodiment, a TeNT binding domain comprising a TeNTH_(CC) binding domain comprises a modification to amino acids 1128-1177,amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1128-1177, amino acids1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8, at least 75% aminoacid identity with amino acids 1128-1177, amino acids 1195-1240, oramino acids 1255-1315 of SEQ ID NO: 8, at least 80% amino acid identitywith amino acids 1128-1177, amino acids 1195-1240, or amino acids1255-1315 of SEQ ID NO: 8, at least 85% amino acid identity with aminoacids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQID NO: 8, at least 90% amino acid identity with amino acids 1128-1177,amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8 or atleast 95% amino acid identity with amino acids 1128-1177, amino acids1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In yet otheraspects of this embodiment, a TeNT H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1128-1177, amino acids 1195-1240,or amino acids 1255-1315 of SEQ ID NO: 8, at most 75% amino acididentity with amino acids 1128-1177, amino acids 1195-1240, or aminoacids 1255-1315 of SEQ ID NO: 8, at most 80% amino acid identity withamino acids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315of SEQ ID NO: 8, at most 85% amino acid identity with amino acids1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO:8, at most 90% amino acid identity with amino acids 1128-1177, aminoacids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8 or at most 95%amino acid identity with amino acids 1128-1177, amino acids 1195-1240,or amino acids 1255-1315 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO:8. In other aspects of this embodiment, a TeNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO:8. In yet other aspects of this embodiment, a TeNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1128-1177,amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. Inother aspects of this embodiment, a TeNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1128-1177,amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. Instill other aspects of this embodiment, a TeNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1128-1177,amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. Inother aspects of this embodiment, a TeNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1128-1177,amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1128-1177,amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. Inother aspects of this embodiment, a TeNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1128-1177,amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In yetother aspects of this embodiment, a TeNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1128-1177, aminoacids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1128-1177, aminoacids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In stillother aspects of this embodiment, a TeNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1128-1177, aminoacids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1128-1177, aminoacids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8.

In another embodiment, a TeNT binding domain comprising a TeNT H_(CC)binding domain comprises a β4/β5 hairpin turn of a β-trefoil domain of aTeNT H_(CC) binding domain or a β8/β9 hairpin turn of a β-trefoil domainof a TeNT H_(CC) binding domain. In another aspect of this embodiment, aTeNT binding domain comprising a TeNT H_(CC) binding domain comprises amodification of amino acids 1178-1194 or amino acids 1241-1254 of SEQ IDNO: 8.

In other aspects of this embodiment, a TeNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1178-1194 or aminoacids 1241-1254 of SEQ ID NO: 8, at least 75% amino acid identity withamino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8, at least80% amino acid identity with amino acids 1178-1194 or amino acids1241-1254 of SEQ ID NO: 8, at least 85% amino acid identity with aminoacids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8, at least 90%amino acid identity with amino acids 1178-1194 or amino acids 1241-1254of SEQ ID NO: 8 or at least 95% amino acid identity with amino acids1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In yet other aspectsof this embodiment, a TeNT H_(CC) binding domain comprising a β-trefoilfold domain comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 1178-1194 or amino acids 1241-1254 of SEQ IDNO: 8, at most 75% amino acid identity with amino acids 1178-1194 oramino acids 1241-1254 of SEQ ID NO: 8, at most 80% amino acid identitywith amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8, atmost 85% amino acid identity with amino acids 1178-1194 or amino acids1241-1254 of SEQ ID NO: 8, at most 90% amino acid identity with aminoacids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8 or at most 95%amino acid identity with amino acids 1178-1194 or amino acids 1241-1254of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In other aspects ofthis embodiment, a TeNT H_(CC) binding domain comprising a β-trefoilfold domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or 10 non-contiguous aminoacid substitutions relative to amino acids 1178-1194 or amino acids1241-1254 of SEQ ID NO: 8. In other aspects of this embodiment, anon-contiguous amino acid substitution of any amino acid from aminoacids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8 can be replacedwith glycine. In other aspects of this embodiment, a non-contiguousamino acid substitution of any hydrophobic amino acid from amino acids1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8 can be replaced withphenylalanine. In yet other aspects of this embodiment, a TeNT H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1178-1194 oramino acids 1241-1254 of SEQ ID NO: 8. In still other aspects of thisembodiment, a TeNT H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidadditions relative to amino acids 1178-1194 or amino acids 1241-1254 ofSEQ ID NO: 8. In other aspects of this embodiment, a TeNT H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine or 10 non-contiguous amino acid additions relative to amino acids1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid substitutions relative to amino acids 1178-1194 oramino acids 1241-1254 of SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to amino acids 1178-1194 or amino acids 1241-1254of SEQ ID NO: 8. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1178-1194 or aminoacids 1241-1254 of SEQ ID NO: 8 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1178-1194 or amino acids1241-1254 of SEQ ID NO: 8 can be replaced with phenylalanine. In yetother aspects of this embodiment, a TeNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid deletions relative to amino acids 1178-1194 oramino acids 1241-1254 of SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino aciddeletions relative to amino acids 1178-1194 or amino acids 1241-1254 ofSEQ ID NO: 8. In still other aspects of this embodiment, a TeNT H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid additions relative to amino acids 1178-1194 or amino acids1241-1254 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(CC) binding domaincomprises a substitution of amino acid Trp 1118, Gly 1119, Leu 1122, Tyr1128, Tyr 1129, Gly 1172, Ile 1177, Asp 1194, Asp 1222, Thr 1270, Ser1287, Trp 1289, Tyr 1290, His 1293, Gly 1300 or Trp 1303, or anycombination thereof, the substitution enhancing the binding activity ofthe TeNT H_(CC) binding domain. In other aspects of this embodiment, aTeNT H_(CC) binding domain comprises a deletion of amino acid Trp 1118,Gly 1119, Leu 1122, Tyr 1128, Tyr 1129, Gly 1172, Ile 1177, Asp 1194,Asp 1222, Thr 1270, Ser 1287, Trp 1289, Tyr 1290, His 1293, Gly 1300 orTrp 1303, or any combination thereof, the deletion enhancing the bindingactivity of the TeNT H_(CC) binding domain.

In another embodiment, a binding domain comprises a BaNT binding domain.In an aspect of this embodiment, a BaNT binding domain comprises a BaNTH_(C) binding domain. In other aspects of this embodiment, a BaNTbinding domain comprising a BaNT H_(C) binding domain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 858-1268 of SEQ ID NO: 9, at least 75% amino acid identity withamino acids 858-1268 of SEQ ID NO: 9, at least 80% amino acid identitywith amino acids 858-1268 of SEQ ID NO: 9, at least 85% amino acididentity with amino acids 858-1268 of SEQ ID NO: 9, at least 90% aminoacid identity with amino acids 858-1268 of SEQ ID NO: 9 or at least 95%amino acid identity with amino acids 858-1268 of SEQ ID NO: 9. In yetother aspects of this embodiment, a BaNT binding domain comprising aBaNT H_(C) binding domain comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 858-1268 of SEQ ID NO: 9, atmost 75% amino acid identity with amino acids 858-1268 of SEQ ID NO: 9,at most 80% amino acid identity with amino acids 858-1268 of SEQ ID NO:9, at most 85% amino acid identity with amino acids 858-1268 of SEQ IDNO: 9, at most 90% amino acid identity with amino acids 858-1268 of SEQID NO: 9 or at most 95% amino acid identity with amino acids 858-1268 ofSEQ ID NO: 9.

In other aspects of this embodiment, a BaNT binding domain comprising aBaNT H_(C) binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100, or 200 non-contiguous amino acid substitutions relative toamino acids 858-1268 of SEQ ID NO: 9. In other aspects of thisembodiment, a BaNT binding domain comprising a BaNT H_(C) binding domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid substitutions relative to amino acids 858-1268of SEQ ID NO: 9. In yet other aspects of this embodiment, a BaNT bindingdomain comprising a BaNT H_(C) binding domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 858-1268 of SEQ ID NO: 9. In other aspects ofthis embodiment, a BaNT binding domain comprising a BaNT H_(C) bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid deletions relative to amino acids 858-1268 ofSEQ ID NO: 9. In still other aspects of this embodiment, a BaNT bindingdomain comprising a BaNT H_(C) binding domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 858-1268 of SEQ ID NO: 9. In other aspects ofthis embodiment, a BaNT binding domain comprising a BaNT H_(C) bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid additions relative to amino acids 858-1268 ofSEQ ID NO: 9.

In other aspects of this embodiment, a BaNT binding domain comprising aBaNT H_(C) binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 contiguous amino acid substitutions relative to aminoacids 858-1268 of SEQ ID NO: 9. In other aspects of this embodiment, aBaNT binding domain comprising a BaNT H_(C) binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 858-1268 of SEQ ID NO: 9. In yetother aspects of this embodiment, a BaNT binding domain comprising aBaNT H_(C) binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 contiguous amino acid deletions relative to amino acids858-1268 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNTbinding domain comprising a BaNT H_(C) binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 858-1268 of SEQ ID NO: 9. In stillother aspects of this embodiment, a BaNT binding domain comprising aBaNT H_(C) binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 contiguous amino acid additions relative to amino acids858-1268 of SEQ ID NO: 9. In other aspects of this embodiment, a BaNTbinding domain comprising a BaNT H_(C) binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 858-1268 of SEQ ID NO: 9.

In another embodiment, a BaNT binding domain comprises a BaNT H_(CC)binding domain. In an aspect of this embodiment, a BaNT binding domaincomprising a BaNT H_(CC) binding domain comprises a modification ofamino acids 1128-1315 of SEQ ID NO: 8. In another aspect of thisembodiment, a BaNT binding domain comprising a BaNT H_(CC) bindingdomain comprises a α-fold motif of a β-trefoil domain of a BaNT H_(CC)binding domain, a β-fold motif of a β-trefoil domain of a BaNT H_(CC)binding domain, or a γ-fold motif of a β-trefoil domain of a BaNT H_(CC)binding domain. In another aspect of this embodiment, a BaNT bindingdomain comprising a BaNT H_(CC) binding domain comprises a modificationto amino acids 1095-1142, amino acids 1162-1207, or amino acids1216-1268 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1095-1142, amino acids1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9, at least 75% aminoacid identity with amino acids 1095-1142, amino acids 1162-1207, oramino acids 1216-1268 of SEQ ID NO: 9, at least 80% amino acid identitywith amino acids 1095-1142, amino acids 1162-1207, or amino acids1216-1268 of SEQ ID NO: 9, at least 85% amino acid identity with aminoacids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQID NO: 9, at least 90% amino acid identity with amino acids 1095-1142,amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9 or atleast 95% amino acid identity with amino acids 1095-1142, amino acids1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In yet otheraspects of this embodiment, a BaNT H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1095-1142, amino acids 1162-1207,or amino acids 1216-1268 of SEQ ID NO: 9, at most 75% amino acididentity with amino acids 1095-1142, amino acids 1162-1207, or aminoacids 1216-1268 of SEQ ID NO: 9, at most 80% amino acid identity withamino acids 1095-1142, amino acids 1162-1207, or amino acids 1216-1268of SEQ ID NO: 9, at most 85% amino acid identity with amino acids1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO:9, at most 90% amino acid identity with amino acids 1095-1142, aminoacids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9 or at most 95%amino acid identity with amino acids 1095-1142, amino acids 1162-1207,or amino acids 1216-1268 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO:9. In other aspects of this embodiment, a BaNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1095-1142, amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO:9. In yet other aspects of this embodiment, a BaNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1095-1142,amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. Inother aspects of this embodiment, a BaNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1095-1142,amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. Instill other aspects of this embodiment, a BaNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1095-1142,amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. Inother aspects of this embodiment, a BaNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1095-1142,amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1095-1142,amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. Inother aspects of this embodiment, a BaNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1095-1142,amino acids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In yetother aspects of this embodiment, a BaNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1095-1142, aminoacids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In otheraspects of this embodiment, a BaNT H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1095-1142, aminoacids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In stillother aspects of this embodiment, a BaNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1095-1142, aminoacids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9. In otheraspects of this embodiment, a BaNT H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1095-1142, aminoacids 1162-1207, or amino acids 1216-1268 of SEQ ID NO: 9.

In another embodiment, a BaNT binding domain comprising a BaNT H_(CC)binding domain comprises a β4/β5 hairpin turn of a β-trefoil domain of aBaNT H_(CC) binding domain or a β8/β9 hairpin turn of a β-trefoil domainof a BaNT H_(CC) binding domain. In another aspect of this embodiment, aBaNT binding domain comprising a BaNT H_(CC) binding domain comprises amodification of amino acids 1143-1161 or amino acids 1208-1215 of SEQ IDNO: 9.

In other aspects of this embodiment, a BaNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1143-1161 or aminoacids 1208-1215 of SEQ ID NO: 9, at least 75% amino acid identity withamino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9, at least80% amino acid identity with amino acids 1143-1161 or amino acids1208-1215 of SEQ ID NO: 9, at least 85% amino acid identity with aminoacids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9, at least 90%amino acid identity with amino acids 1143-1161 or amino acids 1208-1215of SEQ ID NO: 9 or at least 95% amino acid identity with amino acids1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9. In yet other aspectsof this embodiment, a BaNT H_(CC) binding domain comprising a β-trefoilfold domain comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 1143-1161 or amino acids 1208-1215 of SEQ IDNO: 9, at most 75% amino acid identity with amino acids 1143-1161 oramino acids 1208-1215 of SEQ ID NO: 9, at most 80% amino acid identitywith amino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9, atmost 85% amino acid identity with amino acids 1143-1161 or amino acids1208-1215 of SEQ ID NO: 9, at most 90% amino acid identity with aminoacids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9 or at most 95%amino acid identity with amino acids 1143-1161 or amino acids 1208-1215of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9. In other aspects ofthis embodiment, a BaNT H_(CC) binding domain comprising a β-trefoilfold domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or 10 non-contiguous aminoacid substitutions relative to amino acids 1143-1161 or amino acids1208-1215 of SEQ ID NO: 9. In other aspects of this embodiment, anon-contiguous amino acid substitution of any amino acid from aminoacids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9 can be replacedwith glycine. In other aspects of this embodiment, a non-contiguousamino acid substitution of any hydrophobic amino acid from amino acids1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9 can be replaced withphenylalanine. In yet other aspects of this embodiment, a BaNT H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9. In otheraspects of this embodiment, a BaNT H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1143-1161 oramino acids 1208-1215 of SEQ ID NO: 9. In still other aspects of thisembodiment, a BaNT H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidadditions relative to amino acids 1143-1161 or amino acids 1208-1215 ofSEQ ID NO: 9. In other aspects of this embodiment, a BaNT H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine or 10 non-contiguous amino acid additions relative to amino acids1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid substitutions relative to amino acids 1143-1161 oramino acids 1208-1215 of SEQ ID NO: 9. In other aspects of thisembodiment, a BaNT H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to amino acids 1143-1161 or amino acids 1208-1215of SEQ ID NO: 9. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1143-1161 or aminoacids 1208-1215 of SEQ ID NO: 9 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1143-1161 or amino acids1208-1215 of SEQ ID NO: 9 can be replaced with phenylalanine. In yetother aspects of this embodiment, a BaNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid deletions relative to amino acids 1143-1161 oramino acids 1208-1215 of SEQ ID NO: 9. In other aspects of thisembodiment, a BaNT H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino aciddeletions relative to amino acids 1143-1161 or amino acids 1208-1215 ofSEQ ID NO: 9. In still other aspects of this embodiment, a BaNT H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1143-1161 or amino acids 1208-1215 of SEQ ID NO: 9. In otheraspects of this embodiment, a BaNT H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid additions relative to amino acids 1143-1161 or amino acids1208-1215 of SEQ ID NO: 9.

In other aspects of this embodiment, a BaNT H_(CC) binding domaincomprises a substitution of amino acid Trp 1085, Gly 1086, Leu 1089, Tyr1095, Tyr 1096, Gly 1137, Ile 1142, Asp 1161, Ile 1189, Phe 1231, Ser1239, Trp 1241, Tyr 1242, Asn 1245, Gly 1254 or Trp 1257, or anycombination thereof, the substitution enhancing the binding activity ofthe BaNT H_(CC) binding domain. In other aspects of this embodiment, aBaNT H_(CC) binding domain comprises a deletion of amino acid Trp 1085,Gly 1086, Leu 1089, Tyr 1095, Tyr 1096, Gly 1137, Ile 1142, Asp 1161,Ile 1189, Phe 1231, Ser 1239, Trp 1241, Tyr 1242, Asn 1245, Gly 1254 orTrp 1257, or any combination thereof, the deletion enhancing the bindingactivity of the BaNT H_(CC) binding domain.

In another embodiment, a binding domain comprises a BuNT binding domain.In an aspect of this embodiment, a BuNT binding domain comprises a BuNTH_(C) binding domain. In other aspects of this embodiment, a BuNTbinding domain comprising a BuNT H_(C) binding domain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 848-1251 of SEQ ID NO: 10, at least 75% amino acid identity withamino acids 848-1251 of SEQ ID NO: 10, at least 80% amino acid identitywith amino acids 848-1251 of SEQ ID NO: 10, at least 85% amino acididentity with amino acids 848-1251 of SEQ ID NO: 10, at least 90% aminoacid identity with amino acids 848-1251 of SEQ ID NO: 10 or at least 95%amino acid identity with amino acids 848-1251 of SEQ ID NO: 10. In yetother aspects of this embodiment, a BuNT binding domain comprising aBuNT H_(C) binding domain comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 848-1251 of SEQ ID NO: 10, atmost 75% amino acid identity with amino acids 848-1251 of SEQ ID NO: 10,at most 80% amino acid identity with amino acids 848-1251 of SEQ ID NO:10, at most 85% amino acid identity with amino acids 848-1251 of SEQ IDNO: 10, at most 90% amino acid identity with amino acids 848-1251 of SEQID NO: 10 or at most 95% amino acid identity with amino acids 848-1251of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT binding domain comprising aBuNT H_(C) binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100, or 200 non-contiguous amino acid substitutions relative toamino acids 848-1251 of SEQ ID NO: 10. In other aspects of thisembodiment, a BuNT binding domain comprising a BuNT H_(C) binding domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid substitutions relative to amino acids 848-1251of SEQ ID NO: 10. In yet other aspects of this embodiment, a BuNTbinding domain comprising a BuNT H_(C) binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 848-1251 of SEQ ID NO: 10. Inother aspects of this embodiment, a BuNT binding domain comprising aBuNT H_(C) binding domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 non-contiguous amino acid deletions relative to aminoacids 848-1251 of SEQ ID NO: 10. In still other aspects of thisembodiment, a BuNT binding domain comprising a BuNT H_(C) binding domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200non-contiguous amino acid additions relative to amino acids 848-1251 ofSEQ ID NO: 10. In other aspects of this embodiment, a BuNT bindingdomain comprising a BuNT H_(C) binding domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid additionsrelative to amino acids 848-1251 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT binding domain comprising aBuNT H_(C) binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 contiguous amino acid substitutions relative to aminoacids 848-1251 of SEQ ID NO: 10. In other aspects of this embodiment, aBuNT binding domain comprising a BuNT H_(C) binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 848-1251 of SEQ ID NO: 10. In yetother aspects of this embodiment, a BuNT binding domain comprising aBuNT H_(C) binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 contiguous amino acid deletions relative to amino acids848-1251 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNTbinding domain comprising a BuNT H_(C) binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 848-1251 of SEQ ID NO: 10. In stillother aspects of this embodiment, a BuNT binding domain comprising aBuNT H_(C) binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100 or 200 contiguous amino acid additions relative to amino acids848-1251 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNTbinding domain comprising a BuNT H_(C) binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 848-1251 of SEQ ID NO: 10.

In another embodiment, a BuNT binding domain comprises a BuNT H_(CC)binding domain. In an aspect of this embodiment, a BuNT binding domaincomprising a BuNT H_(CC) binding domain comprises a modification ofamino acids 1128-1315 of SEQ ID NO: 8. In another aspect of thisembodiment, a BuNT binding domain comprising a BuNT H_(CC) bindingdomain comprises a α-fold motif of a β-trefoil domain of a BuNT H_(CC)binding domain, a β-fold motif of a β-trefoil domain of a BuNT H_(CC)binding domain, or a γ-fold motif of a β-trefoil domain of a BuNT H_(CC)binding domain. In another aspect of this embodiment, a BuNT bindingdomain comprising a BuNT H_(CC) binding domain comprises a modificationto amino acids 1086-1129, amino acids 1147-1190, or amino acids1198-1251 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1086-1129, amino acids1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10, at least 75% aminoacid identity with amino acids 1086-1129, amino acids 1147-1190, oramino acids 1198-1251 of SEQ ID NO: 10, at least 80% amino acid identitywith amino acids 1086-1129, amino acids 1147-1190, or amino acids1198-1251 of SEQ ID NO: 10, at least 85% amino acid identity with aminoacids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQID NO: 10, at least 90% amino acid identity with amino acids 1086-1129,amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10 or atleast 95% amino acid identity with amino acids 1086-1129, amino acids1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In yet otheraspects of this embodiment, a BuNT H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1086-1129, amino acids 1147-1190,or amino acids 1198-1251 of SEQ ID NO: 10, at most 75% amino acididentity with amino acids 1086-1129, amino acids 1147-1190, or aminoacids 1198-1251 of SEQ ID NO: 10, at most 80% amino acid identity withamino acids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251of SEQ ID NO: 10, at most 85% amino acid identity with amino acids1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO:10, at most 90% amino acid identity with amino acids 1086-1129, aminoacids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10 or at most95% amino acid identity with amino acids 1086-1129, amino acids1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO:10. In other aspects of this embodiment, a BuNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO:10. In yet other aspects of this embodiment, a BuNT H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQID NO: 10. In other aspects of this embodiment, a BuNT H_(CC) bindingdomain comprising a β-trefoil fold domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1086-1129, amino acids 1147-1190, or amino acids 1198-1251 of SEQID NO: 10. In still other aspects of this embodiment, a BuNT H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 1086-1129, amino acids 1147-1190, or amino acids1198-1251 of SEQ ID NO: 10. In other aspects of this embodiment, a BuNTH_(CC) binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 1086-1129, amino acids 1147-1190, or amino acids1198-1251 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1086-1129,amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. Inother aspects of this embodiment, a BuNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1086-1129,amino acids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In yetother aspects of this embodiment, a BuNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1086-1129, aminoacids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In otheraspects of this embodiment, a BuNT H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1086-1129, aminoacids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In stillother aspects of this embodiment, a BuNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1086-1129, aminoacids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10. In otheraspects of this embodiment, a BuNT H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1086-1129, aminoacids 1147-1190, or amino acids 1198-1251 of SEQ ID NO: 10.

In another embodiment, a BuNT binding domain comprising a BuNT H_(CC)binding domain comprises a β4/β5 hairpin turn of a β-trefoil domain of aBuNT H_(CC) binding domain or a β8/β9 hairpin turn of a β-trefoil domainof a BuNT H_(CC) binding domain. In another aspect of this embodiment, aBuNT binding domain comprising a BuNT H_(CC) binding domain comprises amodification of amino acids 1130-1146 or amino acids 1191-1197 of SEQ IDNO: 10.

In other aspects of this embodiment, a BuNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least 70% amino acid identity with amino acids 1130-1146 or aminoacids 1191-1197 of SEQ ID NO: 10, at least 75% amino acid identity withamino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10, atleast 80% amino acid identity with amino acids 1130-1146 or amino acids1191-1197 of SEQ ID NO: 10, at least 85% amino acid identity with aminoacids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10, at least 90%amino acid identity with amino acids 1130-1146 or amino acids 1191-1197of SEQ ID NO: 10 or at least 95% amino acid identity with amino acids1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10. In yet otheraspects of this embodiment, a BuNT H_(CC) binding domain comprising aβ-trefoil fold domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 1130-1146 or amino acids 1191-1197of SEQ ID NO: 10, at most 75% amino acid identity with amino acids1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10, at most 80% aminoacid identity with amino acids 1130-1146 or amino acids 1191-1197 of SEQID NO: 10, at most 85% amino acid identity with amino acids 1130-1146 oramino acids 1191-1197 of SEQ ID NO: 10, at most 90% amino acid identitywith amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10 orat most 95% amino acid identity with amino acids 1130-1146 or aminoacids 1191-1197 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10. In other aspects ofthis embodiment, a BuNT H_(CC) binding domain comprising a β-trefoilfold domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or 10 non-contiguous aminoacid substitutions relative to amino acids 1130-1146 or amino acids1191-1197 of SEQ ID NO: 10. In other aspects of this embodiment, anon-contiguous amino acid substitution of any amino acid from aminoacids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10 can bereplaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a BuNT H_(CC) binding domain comprising a β-trefoil fold domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid deletionsrelative to amino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO:10. In other aspects of this embodiment, a BuNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1130-1146 oramino acids 1191-1197 of SEQ ID NO: 10. In still other aspects of thisembodiment, a BuNT H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidadditions relative to amino acids 1130-1146 or amino acids 1191-1197 ofSEQ ID NO: 10. In other aspects of this embodiment, a BuNT H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid additions relative toamino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid substitutions relative to amino acids 1130-1146 oramino acids 1191-1197 of SEQ ID NO: 10. In other aspects of thisembodiment, a BuNT H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to amino acids 1130-1146 or amino acids 1191-1197of SEQ ID NO: 10. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1130-1146 or aminoacids 1191-1197 of SEQ ID NO: 10 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1130-1146 or amino acids1191-1197 of SEQ ID NO: 10 can be replaced with phenylalanine. In yetother aspects of this embodiment, a BuNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid deletions relative to amino acids 1130-1146 oramino acids 1191-1197 of SEQ ID NO: 10. In other aspects of thisembodiment, a BuNT H_(CC) binding domain comprising a β-trefoil folddomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino aciddeletions relative to amino acids 1130-1146 or amino acids 1191-1197 ofSEQ ID NO: 10. In still other aspects of this embodiment, a BuNT H_(CC)binding domain comprising a β-trefoil fold domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1130-1146 or amino acids 1191-1197 of SEQ ID NO: 10. Inother aspects of this embodiment, a BuNT H_(CC) binding domaincomprising a β-trefoil fold domain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid additions relative to amino acids 1130-1146 oramino acids 1191-1197 of SEQ ID NO: 10.

In other aspects of this embodiment, a BuNT H_(CC) binding domaincomprises a substitution of amino acid Trp 1076, Gly 1077, Leu 1080, Tyr1086, Tyr 1087, Gly 1124, Ile 1129, Asp 1146, Glu 1172, Phe 1213, Ser1221, Trp 1223, Tyr 1224, His 1227, Gly 1236 or Trp 1239, or anycombination thereof, the substitution enhancing the binding activity ofthe BuNT H_(CC) binding domain. In other aspects of this embodiment, aBuNT H_(CC) binding domain comprises a deletion of amino acid Trp 1085,Gly 1086, Leu 1089, Trp 1076, Gly 1077, Leu 1080, Tyr 1086, Tyr 1087,Gly 1124, Ile 1129, Asp 1146, Glu 1172, Phe 1213, Ser 1221, Trp 1223,Tyr 1224, His 1227, Gly 1236 or Trp 1239, or any combination thereof,the deletion enhancing the binding activity of the BuNT H_(CC) bindingdomain.

Another example of a binding domain, includes, without limitation, aClostridial toxin non-toxin associated protein, such as, e.g., non-toxicnon-hemagglutinin (NTNH), hemagglutinin-17 (HA-17), hemagglutinin-33(HA-33) and hemagglutinin-70 (HA-70). In vivo, Clostridial bacteriaproduce a toxin complex comprising the approximately 150-kDa Clostridialtoxin and other proteins collectively called nontoxin associatedproteins (NAPs). Identified NAPs include proteins possessinghemaglutination activity, such, e.g., a hemagglutinin of approximately17-kDa (HA-17), a hemagglutinin of approximately 33-kDa (HA-33) and ahemagglutinin of approximately 70-kDa (HA-70); as well as non-toxicnon-hemagglutinin (NTNH), a protein of approximately 130-kDa, see, e.g.,Eric A. Johnson and Marite Bradshaw, Clostridial botulinum and itsNeurotoxins: A Metabolic and Cellular Perspective, 39 Toxicon 1703-1722(2001); and Stephanie Raffestin et al., Organization and Regulation ofthe Neurotoxin Genes in Clostridium botulinum and Clostridium tetani, 10Anaerobe 93-100 (2004). The toxin complex is important for theintoxication process because it provides protection from adverseenvironmental conditions, resistance to protease digestion, and appearsto facilitate internalization and activation of the toxin.

Recent crystallography experiments have revealed that HA-17, HA-33 andNTNH from various Clostridial bacteria contain a region comprisingβ-trefoil domains very similar to the single β-trefoil domain present inthe binding domain of Clostridial toxins, see, e.g., Kaoru Inoue et al.,Structural Analysis by X-Ray Crystallography and Calorimetry of aHaemagglutinin Component (HA 1) of the Progenitor Toxin from Clostridiumbotulinum, 149 Microbiol. 3361-3370 (2003); and Joseph W. Arndt et al.,The Structure of the Neurotoxin-Associated Protein HA33/A fromClostridial botulinum Suggests a Reoccurring β-trefoil Fold in theProgenitor Toxin Complex, 346 J. Mol. Biol. 1083-1093 (2005). Forexample, HA-33 from Clostridium botulinum serotype A has two β-trefoildomains, each of which consists of three potential carbohydrate bindingmoieties or β-trefoil folds, designated 1α (amino acids 10-55 of SEQ IDNO: 11), 1β (amino acids 56-102 of SEQ ID NO: 11) and 1γ (amino acids103-144 of SEQ ID NO: 11) for the first β-trefoil domain and 2α (aminoacids 151-197 of SEQ ID NO: 11), 2β (amino acids 198-245 of SEQ ID NO:11) and 2γ (amino acids 246-293 of SEQ ID NO: 11) for the secondβ-trefoil domain. Mutations in conserved amino acids of the carbohydratebinding moiety result in a loss of carbohydrate binding, see, e.g.,Kaoru Inoue et al., Structural Analysis by X-Ray Crystallography andCalorimetry of a Haemagglutinin Component (HA1) of the Progenitor Toxinfrom Clostridium botulinum, 149 Microbiol. 3361-3370 (2003).

TABLE 3 β-trefoil Domains of Clostridial HA-33 Proteins Amino AcidSequence Region of Carbohydrate Binding Moieties 1β4/β5 1β8/β9 ProteinSEQ ID NO: 1α-fold β-hairpin turn 1β-fold β-hairpin turn 1γ-foldHA-33/A1 11 10-54 55-59 60-100 101-104 105-144 HA-33/A2 12 10-54 55-5960-100 101-104 105-144 HA-33/A3 13 10-54 55-59 60-100 101-104 105-144HA-33/A4 14 10-56 57-61 62-102 103-106 107-146 HA-33/A5 15 10-54 55-5960-100 101-104 105-144 HA-33/B1 16 10-54 55-59 60-100 101-104 105-144HA-33/B2 17 10-56 57-61 62-102 103-106 107-146 HA-33/C1-1 18 10-54 55-5960-98   99-102 103-141 HA-33/C1-2 19 10-54 55-59 60-98   99-102 103-141HA-33/D1 20 10-54 55-59 60-98   99-102 103-141

These β-trefoil domains are also found in the HA-33 proteins produced byClostridium botulinum serotype B, serotype C1 and serotype D andsequence alignments revealed that amino acids essential for overallcarbohydrate binding are conserved. The amino acids predicted to beessential for carbohydrate binding are as follows Asp 263, Tyr 265, Gln268, Gln 276, Phe 278 and Gln 286 of HA-33/A1 of SEQ ID NO: 11, HA-33/A2of SEQ ID NO: 12, HA-33/A3 of SEQ ID NO: 13, HA-33/A5 of SEQ ID NO: 14and HA-33/B2 of SEQ ID NO: 15; Asp 264, Tyr 266, Gln 269, Gln 277, Phe279 and Gln 287 of HA-33/A4 of SEQ ID NO: 14; Asp 262, Tyr 264, Gln 267,Gln 275, Phe 277 and Gln 285 of HA-33/B1 of SEQ ID NO: 16; Asp 255, Val257, Gly 260, Gln 268, Typ 270 and Gln 278 of HA-33/C1 of SEQ ID NO: 18;and Asp 256, Tyr 258, Gln 261, Ile 269, Asp 271 and Gln 279 of HA-33/C2of SEQ ID NO: 19 and HA-33/D of SEQ ID NO: 20. Immunoaffinity columnchromatography and pull-down assays have shown that HA-33 can bindsynaptotagmin II, a putative Clostridial toxin receptor, see, e.g., YuZhou et al., Haemagglutinin-33 of Type Q Botulinum Neurotoxin ComplexBinds with Synaptotagmin II, 272 FEBS Lett. 2717-2726 (2005). The aminoacid sequences comprising the β-trefoil domains found in variousClostridial HA-33 proteins are shown in Tables 3 and 4.

TABLE 4 β-trefoil Domains of Clostridial HA-33 Proteins Amino AcidSequence Region of Carbohydrate Binding Moieties 2β4/β5 2β8/β9 ProteinSEQ ID NO: 2α-fold β-hairpin turn 2β-fold β-hairpin turn 2γ-foldHA-33/A1 11 151-195 196-199 200-242 243-248 249-293 HA-33/A2 12 151-195196-199 200-242 243-248 249-293 HA-33/A3 13 151-195 196-199 200-242243-248 249-293 HA-33/A4 14 153-197 198-201 202-243 244-249 250-294HA-33/A5 15 151-195 196-199 200-242 243-248 249-279 HA-33/B1 16 151-195196-199 200-241 242-247 248-292 HA-33/B2 17 153-197 198-201 200-242243-248 249-291 HA-33/C1-1 18 148-190 191-194 195-234 235-240 241-285HA-33/C1-2 19 148-190 191-194 195-235 236-241 242-286 HA-33/D1 20148-190 191-194 195-235 236-241 242-286

Further analysis of the β-trefoil domain sequence of HA-33 alsoidentified β-trefoil domains in HA-17 and NTNH, see, e.g., Joseph W.Arndt et al., The Structure of the Neurotoxin-Associated Protein HA33/Afrom Clostridial botulinum Suggests a Reoccurring β-trefoil Fold in theProgenitor Toxin Complex, 346 J. Mol. Biol. 1083-1093 (2005). The HA-17comprises a single β-trefoil domain containing three carbohydratebinding moieties or β-trefoil folds. The carbohydrate binding moietiesof HA-17 exhibits the greatest sequence similarity with the 2γcarbohydrate binding moiety of HA-33. These β-trefoil domains are alsofound in the HA-17 proteins produced by Clostridium botulinum serotypeB, serotype C1 and serotype D and sequence alignments revealed thatamino acids essential for overall carbohydrate binding are conserved.The amino acids predicted to be essential for carbohydrate binding areas follows Tyr 110, Typ 112, Tyr 115, Pro 130, Phe 132 and Asn 138 ofHA-17/A of SEQ ID NO: 21, HA-17/B of SEQ ID NO: 22, HA-17/C1 of SEQ IDNO: 23 and HA-17/D of SEQ ID NO: 24. The amino acid sequences comprisingthe β-trefoil domains found in various Clostridial HA-17 proteins areshown in Table 5.

TABLE 5 β-trefoil Domains of Clostridial HA-17 Proteins Amino AcidSequence Region of Carbohydrate Binding Moieties β4/β5 β8/β9 Protein SEQID NO: α-fold β-hairpin turn β-fold β-hairpin turn γ-fold HA-17/A 219-50 51-54 55-91 92-94 95-146 HA-17/B 22 9-50 51-54 55-91 92-94 95-146HA-17/C1 23 9-50 51-54 55-91 92-94 95-146 HA-17/D 24 9-50 51-54 55-9192-94 95-146

NTNH from various Clostridial bacteria shows significant sequencesimilarity to the β-trefoil domains present in the cell binding domainof BoNT/A and TeNT. The high degree of structural similarity isinteresting in light of the low sequence similarity between NTNH and theClostridial toxins. Furthermore, since NTNH of the various serotypeshave greater sequence similarity than the Clostridial toxins, it islikely that the NTNH produced by other Clostridial strains will alsohave β-trefoil domains exhibiting high structural similarity with thebinding domains of Clostridial toxins. The β-trefoil domains of variousClostridial NTNHs are as follows: amino acids 1050-1193 of NTNH/A1 ofSEQ ID NO: 25; amino acids 1050-1198 of NTNH/A2 of SEQ ID NO: 26; aminoacids 1050-1193 of NTNH/A3 of SEQ ID NO: 27; amino acids 1049-1197 ofNTNH/B of SEQ ID NO: 28; amino acids 1049-1196 of NTNH/C1 of SEQ ID NO:29; amino acids 1049-1196 of NTNH/D of SEQ ID NO: 30; amino acids1014-1162 of NTNH/E of SEQ ID NO: 30; amino acids 1016-1159 of NTNH/F1of SEQ ID NO: 32; amino acids 1017-1165 of NTNH/F2 of SEQ ID NO: 33; andamino acids 1050-1198 of NTNH/G of SEQ ID NO: 34. The amino acidsequences comprising the β-trefoil domains found in various ClostridialNTNH proteins are shown in Table 6.

TABLE 6 β-trefoil Domains of Clostridial NTNH Proteins Amino AcidSequence Region of Carbohydrate Binding Moieties β4/β5 β8/β9 Protein SEQID NO: α-fold β-hairpin turn β-fold β-hairpin turn γ-fold NTNH/A1 251050-1097 1098-1110 1111-1138 1139-1148 1149-1194 NTNH/A2 26 1050-10971098-1110 1111-1139 1140-1148 1149-1199 NTNH/A3 27 1050-1097 1098-11101111-1138 1139-1148 1149-1194 NTNH/B 28 1049-1096 1097-1109 1110-11381139-1147 1148-1198 NTNH/C1 29 1049-1096 1097-1109 1110-1138 1139-11471148-1197 NTNH/D 30 1049-1096 1097-1109 1110-1138 1139-1147 1148-1197NTNH/E 31 1014-1061 1062-1074 1075-1103 1104-1113 1114-1163 NTNH/F1 321016-1063 1064-1076 1077-1104 1105-1114 1115-1160 NTNH/F2 33 1017-10641065-1077 1078-1106 1107-1116 1117-1166 NTNH/G 34 1050-1097 1098-11101111-1139 1140-1149 1150-1199

The β-trefoil domains present in the Clostridial toxin, HA-33, HA-17 andNTNH collectively form nearly half the mass of a Clostridial toxincomplex and underlies the apparent importance of carbohydrate binding inthe cell binding step of the intoxication process. This observation isfurther enhanced by the fact that a Clostridial toxin alone is not aseffective in intoxicating a cell as the entire toxin complex. Onepotential explanation for this enhanced binding activity is thepresence, both in type and in quantity, of the β-trefoil domains presentin HA-33, HA-17 and NTNH. Therefore, the high prediction of structuralsimilarity of the β-trefoil domains present in HA-33, HA-17 and NTNHrelative to the β-trefoil domain found in Clostridial toxins provides apotential source of binding domains useful for developing multivalentClostridial toxins. As a non-limiting example, a carbohydrate bindingmoiety or a β-trefoil fold from HA-33, HA-17 or NTNH can be substitutedfor the naturally occurring carbohydrate binding moiety present in aClostridial toxin. As another non-limiting example, a carbohydratebinding moiety or a β-trefoil fold from HA-33, HA-17 or NTNH can beadded in addition to the naturally occurring carbohydrate binding moietypresent in a Clostridial toxin. As yet another non-limiting example, amultiple carbohydrate binding moieties or a β-trefoil folds from HA-33,HA-17 or NTNH can be substituted for the naturally occurringcarbohydrate binding moiety present in a Clostridial toxin. As stillanother non-limiting example, multiple carbohydrate binding moieties ora β-trefoil folds from HA-33, HA-17 or NTNH can be added in addition tothe naturally occurring carbohydrate binding moiety present in aClostridial toxin. As another non-limiting example, multiplecarbohydrate binding moieties or a β-trefoil folds from a Clostridialtoxin binding domain can be added in addition to the naturally occurringcarbohydrate binding moiety present in a Clostridial toxin.

As used herein, the term “Non-toxin Associated Protein” is synonymouswith “NAP” and means a Clostridial NAP with selective binding activity,such as, e.g., a binding affinity or a binding specificity, for aClostridial toxin receptor. It is envisioned that both naturallyoccurring NAPs as well as non-naturally occurring NAPs can be used as abinding domain. A Clostridial NAP includes, without limitation,naturally occurring Clostridial NAP variants, such as, e.g., ClostridialNAP isoforms and Clostridial NAP subtypes; non-naturally occurringClostridial NAP variants, such as, e.g., conservative Clostridial NAPvariants, non-conservative Clostridial NAP variants, Clostridial NAPchimerics, active Clostridial NAP fragments thereof, or any combinationthereof.

As used herein, the term “Clostridial NAP variant,” whethernaturally-occurring or non-naturally-occurring, means a Clostridial NAPthat has at least one amino acid change from the corresponding region ofthe disclosed reference sequences (see Tables 3-6) and can be describedin percent identity to the corresponding region of that referencesequence. Unless expressly indicated, all Clostridial NAP variantsdisclosed in the present specification are capable of executing the cellbinding step of the intoxication process.

It is recognized by those of skill in the art that within eachClostridial bacterium there can be naturally occurring Clostridial NAPvariants that differ somewhat in their amino acid sequence, and also inthe nucleic acids encoding these proteins. For example, there arepresently five BoNT/A HA-33 variants, HA-33/A1, HA-33/A2, HA-33/A3,HA-33/A4 and HA-33/A5 (Tables 3 and 4), with specific HA-33 variantsshowing various degrees of amino acid divergence when compared toanother HA-33 variant. As another example, there are presently threeBoNT/A NTNH-33 variants, NTNH/A1, NTNH/A2 and NTNH/A3 (Table 6), withspecific NTNH variant showing various degrees of amino acid divergencewhen compared to another NTNH variant. As used herein, the term“naturally occurring Clostridial NAP variant” means any Clostridial NAPproduced by a naturally-occurring process, including, withoutlimitation, Clostridial NAP isoforms produced from alternatively-splicedtranscripts, Clostridial NAP isoforms produced by spontaneous mutationand Clostridial NAP subtypes. A naturally occurring Clostridial NAPvariant can function in substantially the same manner as the referenceClostridial NAP on which the naturally occurring Clostridial NAP variantis based, and can be substituted for the reference Clostridial NAP inany aspect of the present invention. A naturally occurring ClostridialNAP variant may substitute one or more amino acids, two or more aminoacids, three or more amino acids, four or more amino acids, five or moreamino acids, ten or more amino acids, 20 or more amino acids, 30 or moreamino acids, 40 or more amino acids, 50 or more amino acids or 100 ormore amino acids from the reference Clostridial NAP on which thenaturally occurring Clostridial NAP variant is based. A naturallyoccurring Clostridial NAP variant can also substitute at least 10contiguous amino acids, at least 15 contiguous amino acids, at least 20contiguous amino acids, or at least 25 contiguous amino acids from thereference Clostridial NAP on which the naturally occurring ClostridialNAP variant is based, that possess at least 50% amino acid identity, 65%amino acid identity, 75% amino acid identity, 85% amino acid identity or95% amino acid identity to the reference Clostridial NAP on which thenaturally occurring Clostridial NAP variant is based.

A non-limiting examples of a naturally occurring Clostridial NAP variantis a Clostridial NAP isoform such as, e.g., a BoNT/A HA-33 isoform, aClostridial botulinum serotype B HA-33 isoform, a Clostridial botulinumserotype C1 HA-33 isoform, a Clostridial botulinum serotype D HA-33isoform, a BoNT/A HA-17 isoform, a Clostridial botulinum serotype BHA-17 isoform, a Clostridial botulinum serotype C1 HA-17 isoform, aClostridial botulinum serotype D HA-17 isoform, a BoNT/A NTNH isoform, aClostridial botulinum serotype B NTNH isoform, a Clostridial botulinumserotype C1 NTNH isoform, a Clostridial botulinum serotype D NTNHisoform, a Clostridial botulinum serotype E NTNH isoform, a Clostridialbotulinum serotype F NTNH isoform and a Clostridial botulinum serotype GNTNH isoform. A Clostridial NAP isoform can function in substantiallythe same manner as the reference Clostridial NAP on which theClostridial NAP isoform is based, and can be substituted for thereference Clostridial NAP in any aspect of the present invention.

Another non-limiting examples of a naturally occurring Clostridial NAPvariant is a Clostridial NAP subtype such as, e.g., a BoNT/A HA-33subtype, a Clostridial botulinum serotype B HA-33 subtype, a Clostridialbotulinum serotype C1 HA-33 subtype, a Clostridial botulinum serotype DHA-33 subtype, a BoNT/A HA-17 subtype, a Clostridial botulinum serotypeB HA-17 subtype, a Clostridial botulinum serotype C1 HA-17 subtype, aClostridial botulinum serotype D HA-17 subtype, a BoNT/A NTNH subtype, aClostridial botulinum serotype B NTNH subtype, a Clostridial botulinumserotype C1 NTNH subtype, a Clostridial botulinum serotype D NTNHsubtype, a Clostridial botulinum serotype E NTNH subtype, a Clostridialbotulinum serotype F NTNH subtype and a Clostridial botulinum serotype GNTNH subtype. A Clostridial NAP subtype can function in substantiallythe same manner as the reference Clostridial NAP on which theClostridial NAP subtype is based, and can be substituted for thereference Clostridial NAP in any aspect of the present invention.

As used herein, the term “non-naturally occurring Clostridial NAPvariant” means any Clostridial NAP produced with the aid of humanmanipulation, including, without limitation, Clostridial NAPs producedby genetic engineering using random mutagenesis or rational design andClostridial NAPs produced by chemical synthesis. Non-limiting examplesof non-naturally occurring Clostridial NAP variants include, e.g.,conservative Clostridial NAP variants, non-conservative Clostridial NAPvariants, Clostridial NAP chimeric variants and active Clostridial NAPfragments.

As used herein, the term “conservative Clostridial NAP variant” means aClostridial NAP that has at least one amino acid substituted by anotheramino acid or an amino acid analog that has at least one propertysimilar to that of the original amino acid from the referenceClostridial NAP sequence (see Tables 3-6). Examples of propertiesinclude, without limitation, similar size, topography, charge,hydrophobicity, hydrophilicity, lipophilicity, covalent-bondingcapacity, hydrogen-bonding capacity, a physicochemical property, of thelike, or any combination thereof. A conservative Clostridial NAP variantcan function in substantially the same manner as the referenceClostridial NAP on which the conservative Clostridial NAP variant isbased, and can be substituted for the reference Clostridial NAP in anyaspect of the present invention. A conservative Clostridial NAP variantmay substitute one or more amino acids, two or more amino acids, threeor more amino acids, four or more amino acids, five or more amino acids,ten or more amino acids, 20 or more amino acids, 30 or more amino acids,40 or more amino acids or 50 or more amino acids from the referenceClostridial NAP on which the conservative Clostridial NAP variant isbased. A conservative Clostridial NAP variant can also substitute atleast 10 contiguous amino acids, at least 15 contiguous amino acids, atleast 20 contiguous amino acids, or at least 25 contiguous amino acidsfrom the reference Clostridial NAP on which the conservative ClostridialNAP variant is based, that possess at least 50% amino acid identity, 65%amino acid identity, 75% amino acid identity, 85% amino acid identity or95% amino acid identity to the reference Clostridial NAP on which theconservative Clostridial NAP variant is based. Non-limiting examples ofa conservative Clostridial NAP variant include, e.g., a conservativeBoNT/A HA-33 variant, a conservative Clostridial botulinum serotype BHA-33 variant, a conservative Clostridial botulinum serotype C1 HA-33variant, a conservative Clostridial botulinum serotype D HA-33 variant,a conservative BoNT/A HA-17 variant, a conservative Clostridialbotulinum serotype B HA-17 variant, a conservative Clostridial botulinumserotype C1 HA-17 variant, a conservative Clostridial botulinum serotypeD HA-17 variant, a conservative BoNT/A NTNH variant, a conservativeClostridial botulinum serotype B NTNH variant, a conservativeClostridial botulinum serotype C1 NTNH variant, a conservativeClostridial botulinum serotype D NTNH variant, a conservativeClostridial botulinum serotype E NTNH variant, a conservativeClostridial botulinum serotype F NTNH variant and a conservativeClostridial botulinum serotype G NTNH variant.

As used herein, the term “non-conservative Clostridial NAP variant”means a Clostridial NAP in which 1) at least one amino acid is deletedfrom the reference Clostridial NAP on which the non-conservativeClostridial NAP variant is based; 2) at least one amino acid added tothe reference Clostridial NAP on which the non-conservative ClostridialNAP is based; or 3) at least one amino acid is substituted by anotheramino acid or an amino acid analog that does not share any propertysimilar to that of the original amino acid from the referenceClostridial NAP sequence (see Tables 3-6). A non-conservativeClostridial NAP variant can function in substantially the same manner asthe reference Clostridial NAP on which the non-conservative ClostridialNAP variant is based, and can be substituted for the referenceClostridial NAP in any aspect of the present invention. Anon-conservative Clostridial NAP variant can delete one or more aminoacids, two or more amino acids, three or more amino acids, four or moreamino acids, five or more amino acids, and ten or more amino acids fromthe reference Clostridial NAP on which the non-conservative ClostridialNAP variant is based. A non-conservative Clostridial NAP variant can addone or more amino acids, two or more amino acids, three or more aminoacids, four or more amino acids, five or more amino acids, and ten ormore amino acids to the reference Clostridial NAP on which thenon-conservative Clostridial NAP variant is based. A non-conservativeClostridial NAP variant may substitute one or more amino acids, two ormore amino acids, three or more amino acids, four or more amino acids,five or more amino acids, ten or more amino acids, 20 or more aminoacids, 30 or more amino acids, 40 or more amino acids or 50 or moreamino acids from the reference Clostridial NAP on which thenon-conservative Clostridial NAP variant is based. A non-conservativeClostridial NAP variant can also substitute at least 10 contiguous aminoacids, at least 15 contiguous amino acids, at least 20 contiguous aminoacids, or at least 25 contiguous amino acids from the referenceClostridial NAP on which the non-conservative Clostridial NAP variant isbased, that possess at least 50% amino acid identity, 65% amino acididentity, 75% amino acid identity, 85% amino acid identity or 95% aminoacid identity to the reference Clostridial NAP on which thenon-conservative Clostridial NAP variant is based. Non-limiting examplesof a non-conservative Clostridial NAP variant include, e.g., anon-conservative BoNT/A HA-33 variant, a non-conservative Clostridialbotulinum serotype B HA-33 variant, a non-conservative Clostridialbotulinum serotype C1 HA-33 variant, a non-conservative Clostridialbotulinum serotype D HA-33 variant, a non-conservative BoNT/A HA-17variant, a non-conservative Clostridial botulinum serotype B HA-17variant, a non-conservative Clostridial botulinum serotype C1 HA-17variant, a non-conservative Clostridial botulinum serotype D HA-17variant, a non-conservative BoNT/A NTNH variant, a non-conservativeClostridial botulinum serotype B NTNH variant, a non-conservativeClostridial botulinum serotype C1 NTNH variant, a non-conservativeClostridial botulinum serotype D NTNH variant, a non-conservativeClostridial botulinum serotype E NTNH variant, a non-conservativeClostridial botulinum serotype F NTNH variant and a non-conservativeClostridial botulinum serotype G NTNH variant.

As used herein, the term “Clostridial NAP chimeric” means a polypeptidecomprising at least a portion of a Clostridial NAP and at least aportion of at least one other polypeptide to form an enhanced targetingdomain with at least one property different from the referenceClostridial NAP (see Tables 3-6), with the proviso that this ClostridialNAP chimeric can specifically bind to a Clostridial toxin receptorpresent in a Clostridial toxin target cell, and thus participate inexecuting the overall cellular mechanism whereby a Clostridial toxinproteolytically cleaves a substrate.

As used herein, the term “active Clostridial NAP fragment” means any ofa variety of Clostridial NAP fragments comprising the enhanced targetingdomain can be useful in aspects of the present invention with theproviso that these NAP fragments can specifically bind to a Clostridialtoxin receptor present in a Clostridial toxin target cell, and thusparticipate in executing the overall cellular mechanism whereby aClostridial toxin proteolytically cleaves a substrate.

Thus, in an embodiment, a binding domain comprises a NAP. In aspects ofthis embodiment, a NAP comprises a β-trefoil domain derived from a NAP.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a Clostridial HA-33. In an aspect of this embodiment, abinding domain comprising a β-trefoil domain derived from a ClostridialHA-33 comprises, e.g., a β-trefoil domain derived from a BoNT/A HA-33, aβ-trefoil domain derived from a BoNT/B HA-33, a β-trefoil domain derivedfrom a BoNT/C1 HA-33 or a β-trefoil domain derived from a BoNT/D HA-33.In another aspect of this embodiment, a β-trefoil domain derived from aClostridial HA-33 comprises a 1α-fold motif of a β-trefoil domain of aBoNT/A HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/A HA-33, a1γ-fold motif of a β-trefoil domain of a BoNT/A HA-33, a 2α-fold motifof β-trefoil domain of a BoNT/A HA-33, a 2β-fold motif of a β-trefoildomain of a BoNT/A HA-33, or a 2γ-fold motif of a β-trefoil domain of aBoNT/A HA-33. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial HA-33 comprises a 1α-fold motif of aβ-trefoil domain of a BoNT/B HA-33, a 1β-fold motif of a β-trefoildomain of a BoNT/B HA-33, a 1γ-fold motif of a β-trefoil domain of aBoNT/B HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/B HA-33, a2β-fold motif of a β-trefoil domain of a BoNT/B HA-33, or a 2γ-foldmotif of a β-trefoil domain of a BoNT/B HA-33. In another aspect of thisembodiment, a β-trefoil domain derived from a Clostridial HA-33comprises a 1α-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, a1β-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, a 1γ-fold motifof a β-trefoil domain of a BoNT/C1 HA-33, a 2α-fold motif of a β-trefoildomain of a BoNT/C1 HA-33, a 2β-fold motif of a β-trefoil domain of aBoNT/C1 HA-33, or a 2γ-fold motif of a β-trefoil domain of a BoNT/C1HA-33. In another aspect of this embodiment, a β-trefoil domain derivedfrom a Clostridial HA-33 comprises a 1α-fold motif of a β-trefoil domainof a BoNT/D HA-33, a 1β-fold motif of a β-trefoil domain of a BoNT/DHA-33, a 1γ-fold motif of a β-trefoil domain of a BoNT/D HA-33, a2α-fold motif of a β-trefoil domain of a BoNT/D HA-33, a 2β-fold motifof a β-trefoil domain of a BoNT/D HA-33, or a 2γ-fold motif of aβ-trefoil domain of a BoNT/D HA-33.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/A HA-33 of SEQ ID NO: 11. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/A HA-33comprises amino acids 10-144 or amino acids 151-293 of SEQ ID NO: 11. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/A HA-33 comprises a 1α-fold motifof a β-trefoil domain of a BoNT/A HA-33, a 1β-fold motif of a β-trefoildomain of a BoNT/A HA-33, a 1γ-fold motif of a β-trefoil domain of aBoNT/A HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a2β-fold motif of a β-trefoil domain of a BoNT/A HA-33, or a 2γ-foldmotif of a β-trefoil domain of a BoNT/A HA-33 of SEQ ID NO: 11. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/A HA-33 comprises amino acids10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195,amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 11, at least 75% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 11, at least 80% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, atleast 85% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 11, at least 90% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 11 or at least 95% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11.In yet other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 10-54, amino acids 60-100,amino acids 105-144, amino acids 151-195, amino acids 200-242, or aminoacids 249-293 of SEQ ID NO: 11, at most 75% amino acid identity withamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11,at most 80% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 11, at most 85% amino acid identitywith amino acids 10-54, amino acids 60-100, amino acids 105-144, aminoacids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO:11, at most 90% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 11 or at most 95% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 11.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-54,amino acids 60-100, amino acids 105-144, amino acids 151-195, aminoacids 200-242, or amino acids 249-293 of SEQ ID NO: 11. In other aspectsof this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 11. In yet other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 11. In other aspects of this embodiment, a binding domaincomprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11.In still other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 11. In other aspects ofthis embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 11.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 11. In other aspects ofthis embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 11. In yet other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11.In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 11. In still other aspectsof this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 11. In other aspects of this embodiment, a bindingdomain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11.

In another embodiment, a binding domain comprising a BoNT/A HA-33β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domainof a BoNT/A HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of aBoNT/A HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/AHA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-33of SEQ ID NO: 11. In another aspect of this embodiment, a binding domaincomprising a BoNT/A HA-33 β-trefoil domain comprises amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 11.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11,at least 75% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11,at least 80% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11,at least 85% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11,at least 90% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11 orat least 95% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11.In yet other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11,at most 75% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11,at most 80% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11,at most 85% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11,at most 90% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11 orat most 95% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 101-104, amino acids 196-199,or amino acids 243-248 of SEQ ID NO: 11. In other aspects of thisembodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 11. In other aspects of this embodiment, a non-contiguous aminoacid substitution of any amino acid from amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 11 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a BoNT/A HA-33 comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11. In otheraspects of this embodiment, a binding domain comprising a BoNT/A HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 55-59, amino acids 101-104, amino acids 196-199, or amino acids243-248 of SEQ ID NO: 11. In still other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid additions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11. In otheraspects of this embodiment, a binding domain comprising a BoNT/A HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 55-59, amino acids 101-104, amino acids 196-199, or amino acids243-248 of SEQ ID NO: 11.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 101-104, amino acids 196-199,or amino acids 243-248 of SEQ ID NO: 11. In other aspects of thisembodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:11. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11can be replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 11 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid deletions relative toamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 11. In other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid deletions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11. In stillother aspects of this embodiment, a binding domain comprising a BoNT/AHA-33 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 11. In other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid additions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/A HA-33 of SEQ ID NO: 12. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/A HA-33comprises amino acids 10-144 or amino acids 151-293 of SEQ ID NO: 12. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/A HA-33 comprises a 1α-fold motifof a β-trefoil domain of a BoNT/A HA-33, a 1β-fold motif of a β-trefoildomain of a BoNT/A HA-33, a 1γ-fold motif of a β-trefoil domain of aBoNT/A HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a2β-fold motif of a β-trefoil domain of a BoNT/A HA-33, or a 2γ-foldmotif of a β-trefoil domain of a BoNT/A HA-33 of SEQ ID NO: 12. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/A HA-33 comprises amino acids10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195,amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 12, at least 75% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 12, at least 80% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, atleast 85% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 12, at least 90% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 12 or at least 95% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12.In yet other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 10-54, amino acids 60-100,amino acids 105-144, amino acids 151-195, amino acids 200-242, or aminoacids 249-293 of SEQ ID NO: 12, at most 75% amino acid identity withamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12,at most 80% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 12, at most 85% amino acid identitywith amino acids 10-54, amino acids 60-100, amino acids 105-144, aminoacids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO:12, at most 90% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 12 or at most 95% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 12.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-54,amino acids 60-100, amino acids 105-144, amino acids 151-195, aminoacids 200-242, or amino acids 249-293 of SEQ ID NO: 12. In other aspectsof this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 12. In yet other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 12. In other aspects of this embodiment, a binding domaincomprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12.In still other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 12. In other aspects ofthis embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 12.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 12. In other aspects ofthis embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 12. In yet other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12.In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 12. In still other aspectsof this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 12. In other aspects of this embodiment, a bindingdomain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 12.

In another embodiment, a binding domain comprising a BoNT/A HA-33β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domainof a BoNT/A HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of aBoNT/A HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/AHA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-33of SEQ ID NO: 12. In another aspect of this embodiment, a binding domaincomprising a BoNT/A HA-33 β-trefoil domain comprises amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 12.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12,at least 75% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12,at least 80% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12,at least 85% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12,at least 90% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12 orat least 95% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12.In yet other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12,at most 75% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12,at most 80% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12,at most 85% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12,at most 90% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12 orat most 95% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 101-104, amino acids 196-199,or amino acids 243-248 of SEQ ID NO: 12. In other aspects of thisembodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 12. In other aspects of this embodiment, a non-contiguous aminoacid substitution of any amino acid from amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 12 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a BoNT/A HA-33 comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12. In otheraspects of this embodiment, a binding domain comprising a BoNT/A HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 55-59, amino acids 101-104, amino acids 196-199, or amino acids243-248 of SEQ ID NO: 12. In still other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid additions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12. In otheraspects of this embodiment, a binding domain comprising a BoNT/A HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 55-59, amino acids 101-104, amino acids 196-199, or amino acids243-248 of SEQ ID NO: 12.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 101-104, amino acids 196-199,or amino acids 243-248 of SEQ ID NO: 12. In other aspects of thisembodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:12. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12can be replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 12 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid deletions relative toamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 12. In other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid deletions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12. In stillother aspects of this embodiment, a binding domain comprising a BoNT/AHA-33 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 12. In other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid additions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 12.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/A HA-33 of SEQ ID NO: 13. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/A HA-33comprises amino acids 10-144 or amino acids 151-293 of SEQ ID NO: 13. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/A HA-33 comprises a 1α-fold motifof a β-trefoil domain of a BoNT/A HA-33, a 1β-fold motif of a β-trefoildomain of a BoNT/A HA-33, a 1γ-fold motif of a β-trefoil domain of aBoNT/A HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a2β-fold motif of a β-trefoil domain of a BoNT/A HA-33, or a 2γ-foldmotif of a β-trefoil domain of a BoNT/A HA-33 of SEQ ID NO: 13. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/A HA-33 comprises amino acids10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195,amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 13, at least 75% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 13, at least 80% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, atleast 85% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 13, at least 90% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 13 or at least 95% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13.In yet other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 10-54, amino acids 60-100,amino acids 105-144, amino acids 151-195, amino acids 200-242, or aminoacids 249-293 of SEQ ID NO: 13, at most 75% amino acid identity withamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13,at most 80% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 13, at most 85% amino acid identitywith amino acids 10-54, amino acids 60-100, amino acids 105-144, aminoacids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO:13, at most 90% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 13 or at most 95% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 13.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-54,amino acids 60-100, amino acids 105-144, amino acids 151-195, aminoacids 200-242, or amino acids 249-293 of SEQ ID NO: 13. In other aspectsof this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 13. In yet other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 13. In other aspects of this embodiment, a binding domaincomprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13.In still other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 13. In other aspects ofthis embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 13.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 13. In other aspects ofthis embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 13. In yet other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13.In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 13. In still other aspectsof this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 13. In other aspects of this embodiment, a bindingdomain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 13.

In another embodiment, a binding domain comprising a BoNT/A HA-33β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domainof a BoNT/A HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of aBoNT/A HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/AHA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-33of SEQ ID NO: 13. In another aspect of this embodiment, a binding domaincomprising a BoNT/A HA-33 β-trefoil domain comprises amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 13.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13,at least 75% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13,at least 80% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13,at least 85% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13,at least 90% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13 orat least 95% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13.In yet other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13,at most 75% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13,at most 80% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13,at most 85% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13,at most 90% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13 orat most 95% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 101-104, amino acids 196-199,or amino acids 243-248 of SEQ ID NO: 13. In other aspects of thisembodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 13. In other aspects of this embodiment, a non-contiguous aminoacid substitution of any amino acid from amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 13 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a BoNT/A HA-33 comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13. In otheraspects of this embodiment, a binding domain comprising a BoNT/A HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 55-59, amino acids 101-104, amino acids 196-199, or amino acids243-248 of SEQ ID NO: 13. In still other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid additions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13. In otheraspects of this embodiment, a binding domain comprising a BoNT/A HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 55-59, amino acids 101-104, amino acids 196-199, or amino acids243-248 of SEQ ID NO: 13.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 101-104, amino acids 196-199,or amino acids 243-248 of SEQ ID NO: 13. In other aspects of thisembodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:13. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13can be replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 13 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid deletions relative toamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 13. In other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid deletions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13. In stillother aspects of this embodiment, a binding domain comprising a BoNT/AHA-33 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 13. In other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid additions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/A HA-33 of SEQ ID NO: 14. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/A HA-33comprises amino acids 10-146 or amino acids 153-294 of SEQ ID NO: 14. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/A HA-33 comprises a 1α-fold motifof a β-trefoil domain of a BoNT/A HA-33, a 1β-fold motif of a β-trefoildomain of a BoNT/A HA-33, a 1γ-fold motif of a β-trefoil domain of aBoNT/A HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a2β-fold motif of a β-trefoil domain of a BoNT/A HA-33, or a 2γ-foldmotif of a β-trefoil domain of a BoNT/A HA-33 of SEQ ID NO: 14. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/A HA-33 comprises amino acids10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197,amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243,or amino acids 250-294 of SEQ ID NO: 14, at least 75% amino acididentity with amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 202-243, or amino acids250-294 of SEQ ID NO: 14, at least 80% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, atleast 85% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243,or amino acids 250-294 of SEQ ID NO: 14, at least 90% amino acididentity with amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 202-243, or amino acids250-294 of SEQ ID NO: 14 or at least 95% amino acid identity with aminoacids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14.In yet other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 10-56, amino acids 62-102,amino acids 107-146, amino acids 153-197, amino acids 202-243, or aminoacids 250-294 of SEQ ID NO: 14, at most 75% amino acid identity withamino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14,at most 80% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243,or amino acids 250-294 of SEQ ID NO: 14, at most 85% amino acid identitywith amino acids 10-56, amino acids 62-102, amino acids 107-146, aminoacids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO:14, at most 90% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243,or amino acids 250-294 of SEQ ID NO: 14 or at most 95% amino acididentity with amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 202-243, or amino acids250-294 of SEQ ID NO: 14.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-56,amino acids 62-102, amino acids 107-146, amino acids 153-197, aminoacids 202-243, or amino acids 250-294 of SEQ ID NO: 14. In other aspectsof this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-56, amino acids 62-102, aminoacids 107-146, amino acids 153-197, amino acids 202-243, or amino acids250-294 of SEQ ID NO: 14. In yet other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-56, amino acids 62-102, amino acids 107-146,amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQID NO: 14. In other aspects of this embodiment, a binding domaincomprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14.In still other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-56, aminoacids 62-102, amino acids 107-146, amino acids 153-197, amino acids202-243, or amino acids 250-294 of SEQ ID NO: 14. In other aspects ofthis embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 202-243, or amino acids250-294 of SEQ ID NO: 14.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-56, aminoacids 62-102, amino acids 107-146, amino acids 153-197, amino acids202-243, or amino acids 250-294 of SEQ ID NO: 14. In other aspects ofthis embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-56, amino acids 62-102, aminoacids 107-146, amino acids 153-197, amino acids 202-243, or amino acids250-294 of SEQ ID NO: 14. In yet other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14.In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 10-56, aminoacids 62-102, amino acids 107-146, amino acids 153-197, amino acids202-243, or amino acids 250-294 of SEQ ID NO: 14. In still other aspectsof this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 202-243, or amino acids250-294 of SEQ ID NO: 14. In other aspects of this embodiment, a bindingdomain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 14.

In another embodiment, a binding domain comprising a BoNT/A HA-33β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domainof a BoNT/A HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of aBoNT/A HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/AHA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-33of SEQ ID NO: 14. In another aspect of this embodiment, a binding domaincomprising a BoNT/A HA-33 β-trefoil domain comprises amino acids 57-61,amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQID NO: 14.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14,at least 75% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14,at least 80% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14,at least 85% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14,at least 90% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14 orat least 95% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14.In yet other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14,at most 75% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14,at most 80% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14,at most 85% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14,at most 90% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14 orat most 95% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 57-61, amino acids 103-106, amino acids 198-201,or amino acids 244-249 of SEQ ID NO: 14. In other aspects of thisembodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 57-61,amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQID NO: 14. In other aspects of this embodiment, a non-contiguous aminoacid substitution of any amino acid from amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 57-61, amino acids 103-106, amino acids 198-201, oramino acids 244-249 of SEQ ID NO: 14 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a BoNT/A HA-33 comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 57-61, amino acids 103-106,amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14. In otheraspects of this embodiment, a binding domain comprising a BoNT/A HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 57-61, amino acids 103-106, amino acids 198-201, or amino acids244-249 of SEQ ID NO: 14. In still other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid additions relative to amino acids 57-61, amino acids 103-106,amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14. In otheraspects of this embodiment, a binding domain comprising a BoNT/A HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 57-61, amino acids 103-106, amino acids 198-201, or amino acids244-249 of SEQ ID NO: 14.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 57-61, amino acids 103-106, amino acids 198-201,or amino acids 244-249 of SEQ ID NO: 14. In other aspects of thisembodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 57-61, aminoacids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO:14. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14can be replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 57-61, amino acids 103-106, amino acids 198-201, or aminoacids 244-249 of SEQ ID NO: 14 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid deletions relative toamino acids 57-61, amino acids 103-106, amino acids 198-201, or aminoacids 244-249 of SEQ ID NO: 14. In other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid deletions relative to amino acids 57-61, amino acids 103-106,amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14. In stillother aspects of this embodiment, a binding domain comprising a BoNT/AHA-33 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 57-61, amino acids 103-106, amino acids 198-201, or aminoacids 244-249 of SEQ ID NO: 14. In other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid additions relative to amino acids 57-61, amino acids 103-106,amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 14.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/A HA-33 of SEQ ID NO: 15. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/A HA-33comprises amino acids 10-144 or amino acids 151-279 of SEQ ID NO: 15. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/A HA-33 comprises a 1α-fold motifof a β-trefoil domain of a BoNT/A HA-33, a 1β-fold motif of a β-trefoildomain of a BoNT/A HA-33, a 1γ-fold motif of a β-trefoil domain of aBoNT/A HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/A HA-33, a2β-fold motif of a β-trefoil domain of a BoNT/A HA-33, or a 2γ-foldmotif of a β-trefoil domain of a BoNT/A HA-33 of SEQ ID NO: 15. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/A HA-33 comprises amino acids10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195,amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 15, at least 75% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 15, at least 80% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, atleast 85% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 15, at least 90% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 15 or at least 95% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15.In yet other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 10-54, amino acids 60-100,amino acids 105-144, amino acids 151-195, amino acids 200-242, or aminoacids 249-293 of SEQ ID NO: 15, at most 75% amino acid identity withamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15,at most 80% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 15, at most 85% amino acid identitywith amino acids 10-54, amino acids 60-100, amino acids 105-144, aminoacids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO:15, at most 90% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 15 or at most 95% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 15.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-54,amino acids 60-100, amino acids 105-144, amino acids 151-195, aminoacids 200-242, or amino acids 249-293 of SEQ ID NO: 15. In other aspectsof this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 15. In yet other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 15. In other aspects of this embodiment, a binding domaincomprising a BoNT/A HA-33 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15.In still other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 15. In other aspects ofthis embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 15.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 15. In other aspects ofthis embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 15. In yet other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15.In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 15. In still other aspectsof this embodiment, a binding domain comprising a BoNT/A HA-33 β-trefoildomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 15. In other aspects of this embodiment, a bindingdomain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 15.

In another embodiment, a binding domain comprising a BoNT/A HA-33β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domainof a BoNT/A HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of aBoNT/A HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/AHA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A HA-33of SEQ ID NO: 15. In another aspect of this embodiment, a binding domaincomprising a BoNT/A HA-33 β-trefoil domain comprises amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 15.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15,at least 75% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15,at least 80% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15,at least 85% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15,at least 90% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15 orat least 95% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15.In yet other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15,at most 75% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15,at most 80% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15,at most 85% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15,at most 90% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15 orat most 95% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 101-104, amino acids 196-199,or amino acids 243-248 of SEQ ID NO: 15. In other aspects of thisembodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 15. In other aspects of this embodiment, a non-contiguous aminoacid substitution of any amino acid from amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 15 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a BoNT/A HA-33 comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15. In otheraspects of this embodiment, a binding domain comprising a BoNT/A HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 55-59, amino acids 101-104, amino acids 196-199, or amino acids243-248 of SEQ ID NO: 15. In still other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid additions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15. In otheraspects of this embodiment, a binding domain comprising a BoNT/A HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 55-59, amino acids 101-104, amino acids 196-199, or amino acids243-248 of SEQ ID NO: 15.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 101-104, amino acids 196-199,or amino acids 243-248 of SEQ ID NO: 15. In other aspects of thisembodiment, a binding domain comprising a BoNT/A HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:15. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15can be replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 15 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a binding domain comprising aBoNT/A HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid deletions relative toamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 15. In other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid deletions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15. In stillother aspects of this embodiment, a binding domain comprising a BoNT/AHA-33 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 15. In other aspects of this embodiment, abinding domain comprising a BoNT/A HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid additions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 15.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/B HA-33 of SEQ ID NO: 16. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/B HA-33comprises amino acids 10-144 or amino acids 151-292 of SEQ ID NO: 16. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/B HA-33 comprises a 1α-fold motifof a β-trefoil domain of a BoNT/B HA-33, a 1β-fold motif of a β-trefoildomain of a BoNT/B HA-33, a 1γ-fold motif of a β-trefoil domain of aBoNT/B HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/B HA-33, a2β-fold motif of a β-trefoil domain of a BoNT/B HA-33, or a 2γ-foldmotif of a β-trefoil domain of a BoNT/B HA-33 of SEQ ID NO: 16. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/B HA-33 comprises amino acids10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195,amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241,or amino acids 248-292 of SEQ ID NO: 16, at least 75% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-241, or amino acids248-292 of SEQ ID NO: 16, at least 80% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, atleast 85% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241,or amino acids 248-292 of SEQ ID NO: 16, at least 90% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-241, or amino acids248-292 of SEQ ID NO: 16 or at least 95% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16.In yet other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 10-54, amino acids 60-100,amino acids 105-144, amino acids 151-195, amino acids 200-241, or aminoacids 248-292 of SEQ ID NO: 16, at most 75% amino acid identity withamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16,at most 80% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241,or amino acids 248-292 of SEQ ID NO: 16, at most 85% amino acid identitywith amino acids 10-54, amino acids 60-100, amino acids 105-144, aminoacids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO:16, at most 90% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241,or amino acids 248-292 of SEQ ID NO: 16 or at most 95% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-241, or amino acids248-292 of SEQ ID NO: 16.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-54,amino acids 60-100, amino acids 105-144, amino acids 151-195, aminoacids 200-241, or amino acids 248-292 of SEQ ID NO: 16. In other aspectsof this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-241, or amino acids248-292 of SEQ ID NO: 16. In yet other aspects of this embodiment, abinding domain comprising a BoNT/B HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQID NO: 16. In other aspects of this embodiment, a binding domaincomprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16.In still other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-241, or amino acids 248-292 of SEQ ID NO: 16. In other aspects ofthis embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-241, or amino acids248-292 of SEQ ID NO: 16.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-241, or amino acids 248-292 of SEQ ID NO: 16. In other aspects ofthis embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-241, or amino acids248-292 of SEQ ID NO: 16. In yet other aspects of this embodiment, abinding domain comprising a BoNT/B HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16.In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-241, or amino acids 248-292 of SEQ ID NO: 16. In still other aspectsof this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoildomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-241, or amino acids248-292 of SEQ ID NO: 16. In other aspects of this embodiment, a bindingdomain comprising a BoNT/B HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 16.

In another embodiment, a binding domain comprising a BoNT/B HA-33β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domainof a BoNT/B HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of aBoNT/B HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/BHA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/B HA-33of SEQ ID NO: 16. In another aspect of this embodiment, a binding domaincomprising a BoNT/B HA-33 β-trefoil domain comprises amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQID NO: 16.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16,at least 75% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16,at least 80% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16,at least 85% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16,at least 90% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16 orat least 95% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16.In yet other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16,at most 75% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16,at most 80% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16,at most 85% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16,at most 90% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16 orat most 95% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 101-104, amino acids 196-199,or amino acids 242-247 of SEQ ID NO: 16. In other aspects of thisembodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQID NO: 16. In other aspects of this embodiment, a non-contiguous aminoacid substitution of any amino acid from amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 242-247 of SEQ ID NO: 16 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a BoNT/B HA-33 comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16. In otheraspects of this embodiment, a binding domain comprising a BoNT/B HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 55-59, amino acids 101-104, amino acids 196-199, or amino acids242-247 of SEQ ID NO: 16. In still other aspects of this embodiment, abinding domain comprising a BoNT/B HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid additions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16. In otheraspects of this embodiment, a binding domain comprising a BoNT/B HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 55-59, amino acids 101-104, amino acids 196-199, or amino acids242-247 of SEQ ID NO: 16.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 101-104, amino acids 196-199,or amino acids 242-247 of SEQ ID NO: 16. In other aspects of thisembodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO:16. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16can be replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 242-247 of SEQ ID NO: 16 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid deletions relative toamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 242-247 of SEQ ID NO: 16. In other aspects of this embodiment, abinding domain comprising a BoNT/B HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid deletions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16. In stillother aspects of this embodiment, a binding domain comprising a BoNT/BHA-33 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 242-247 of SEQ ID NO: 16. In other aspects of this embodiment, abinding domain comprising a BoNT/B HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid additions relative to amino acids 55-59, amino acids 101-104,amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 16.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/B HA-33 of SEQ ID NO: 17. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/B HA-33comprises amino acids 10-146 or amino acids 153-291 of SEQ ID NO: 17. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/B HA-33 comprises a 1α-fold motifof a β-trefoil domain of a BoNT/B HA-33, a 1β-fold motif of a β-trefoildomain of a BoNT/B HA-33, a 1γ-fold motif of a β-trefoil domain of aBoNT/B HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/B HA-33, a2β-fold motif of a β-trefoil domain of a BoNT/B HA-33, or a 2γ-foldmotif of a β-trefoil domain of a BoNT/B HA-33 of SEQ ID NO: 17. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/B HA-33 comprises amino acids10-56, amino acids 62-102, amino acids 107-146, amino acids 153-197,amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242,or amino acids 249-291 of SEQ ID NO: 17, at least 75% amino acididentity with amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 200-242, or amino acids249-291 of SEQ ID NO: 17, at least 80% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, atleast 85% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242,or amino acids 249-291 of SEQ ID NO: 17, at least 90% amino acididentity with amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 200-242, or amino acids249-291 of SEQ ID NO: 17 or at least 95% amino acid identity with aminoacids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17.In yet other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 10-56, amino acids 62-102,amino acids 107-146, amino acids 153-197, amino acids 200-242, or aminoacids 249-291 of SEQ ID NO: 17, at most 75% amino acid identity withamino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17,at most 80% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242,or amino acids 249-291 of SEQ ID NO: 17, at most 85% amino acid identitywith amino acids 10-56, amino acids 62-102, amino acids 107-146, aminoacids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO:17, at most 90% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242,or amino acids 249-291 of SEQ ID NO: 17 or at most 95% amino acididentity with amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 200-242, or amino acids249-291 of SEQ ID NO: 17.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-56,amino acids 62-102, amino acids 107-146, amino acids 153-197, aminoacids 200-242, or amino acids 249-291 of SEQ ID NO: 17. In other aspectsof this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-56, amino acids 62-102, aminoacids 107-146, amino acids 153-197, amino acids 200-242, or amino acids249-291 of SEQ ID NO: 17. In yet other aspects of this embodiment, abinding domain comprising a BoNT/B HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-56, amino acids 62-102, amino acids 107-146,amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQID NO: 17. In other aspects of this embodiment, a binding domaincomprising a BoNT/B HA-33 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17.In still other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-56, aminoacids 62-102, amino acids 107-146, amino acids 153-197, amino acids200-242, or amino acids 249-291 of SEQ ID NO: 17. In other aspects ofthis embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 200-242, or amino acids249-291 of SEQ ID NO: 17.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-56, aminoacids 62-102, amino acids 107-146, amino acids 153-197, amino acids200-242, or amino acids 249-291 of SEQ ID NO: 17. In other aspects ofthis embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-56, amino acids 62-102, aminoacids 107-146, amino acids 153-197, amino acids 200-242, or amino acids249-291 of SEQ ID NO: 17. In yet other aspects of this embodiment, abinding domain comprising a BoNT/B HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17.In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 10-56, aminoacids 62-102, amino acids 107-146, amino acids 153-197, amino acids200-242, or amino acids 249-291 of SEQ ID NO: 17. In still other aspectsof this embodiment, a binding domain comprising a BoNT/B HA-33 β-trefoildomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 200-242, or amino acids249-291 of SEQ ID NO: 17. In other aspects of this embodiment, a bindingdomain comprising a BoNT/B HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 17.

In another embodiment, a binding domain comprising a BoNT/B HA-33β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domainof a BoNT/B HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of aBoNT/B HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/BHA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/B HA-33of SEQ ID NO: 17. In another aspect of this embodiment, a binding domaincomprising a BoNT/B HA-33 β-trefoil domain comprises amino acids 56-61,amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQID NO: 17.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17,at least 75% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17,at least 80% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17,at least 85% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17,at least 90% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17 orat least 95% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17.In yet other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17,at most 75% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17,at most 80% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17,at most 85% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17,at most 90% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17 orat most 95% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 56-61, amino acids 103-106, amino acids 198-201,or amino acids 243-248 of SEQ ID NO: 17. In other aspects of thisembodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 56-61,amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQID NO: 17. In other aspects of this embodiment, a non-contiguous aminoacid substitution of any amino acid from amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 56-61, amino acids 103-106, amino acids 198-201, oramino acids 243-248 of SEQ ID NO: 17 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a BoNT/B HA-33 comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 56-61, amino acids 103-106,amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17. In otheraspects of this embodiment, a binding domain comprising a BoNT/B HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 56-61, amino acids 103-106, amino acids 198-201, or amino acids243-248 of SEQ ID NO: 17. In still other aspects of this embodiment, abinding domain comprising a BoNT/B HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid additions relative to amino acids 56-61, amino acids 103-106,amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17. In otheraspects of this embodiment, a binding domain comprising a BoNT/B HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 56-61, amino acids 103-106, amino acids 198-201, or amino acids243-248 of SEQ ID NO: 17.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 56-61, amino acids 103-106, amino acids 198-201,or amino acids 243-248 of SEQ ID NO: 17. In other aspects of thisembodiment, a binding domain comprising a BoNT/B HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 56-61, aminoacids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO:17. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17can be replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 56-61, amino acids 103-106, amino acids 198-201, or aminoacids 243-248 of SEQ ID NO: 17 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a binding domain comprising aBoNT/B HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid deletions relative toamino acids 56-61, amino acids 103-106, amino acids 198-201, or aminoacids 243-248 of SEQ ID NO: 17. In other aspects of this embodiment, abinding domain comprising a BoNT/B HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid deletions relative to amino acids 56-61, amino acids 103-106,amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17. In stillother aspects of this embodiment, a binding domain comprising a BoNT/BHA-33 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 56-61, amino acids 103-106, amino acids 198-201, or aminoacids 243-248 of SEQ ID NO: 17. In other aspects of this embodiment, abinding domain comprising a BoNT/B HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid additions relative to amino acids 56-61, amino acids 103-106,amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 17.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/C1 HA-33 of SEQ ID NO: 18. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/C1HA-33 comprises amino acids 10-141 or amino acids 148-285 of SEQ ID NO:18. In another aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/C1 HA-33 comprises a 1α-fold motifof a β-trefoil domain of a BoNT/C1 HA-33, a 1β-fold motif of a β-trefoildomain of a BoNT/C1 HA-33, a 1γ-fold motif of a β-trefoil domain of aBoNT/C1 HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/C1 HA-33,a 2β-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, or a 2γ-foldmotif of a β-trefoil domain of a BoNT/C1 HA-33 of SEQ ID NO: 18. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/C1 HA-33 comprises amino acids10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190,amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 10-54, amino acids 60-98,amino acids 103-141, amino acids 148-190, amino acids 195-234, or aminoacids 241-285 of SEQ ID NO: 18, at least 75% amino acid identity withamino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18,at least 80% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 9, at least 85% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO:18, at least 90% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, oramino acids 241-285 of SEQ ID NO: 18 or at least 95% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO:18. In yet other aspects of this embodiment, a binding domain comprisinga BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, oramino acids 241-285 of SEQ ID NO: 18, at most 75% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO:18, at most 80% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, oramino acids 241-285 of SEQ ID NO: 18, at most 85% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO:18, at most 90% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, oramino acids 241-285 of SEQ ID NO: 18 or at most 95% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO:18.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-54,amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-234, or amino acids 241-285 of SEQ ID NO: 18. In other aspects ofthis embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-234, or amino acids241-285 of SEQ ID NO: 18. In yet other aspects of this embodiment, abinding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-98, amino acids 103-141,amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQID NO: 18. In other aspects of this embodiment, a binding domaincomprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18.In still other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-234, or amino acids 241-285 of SEQ ID NO: 18. In other aspects ofthis embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 10-54, amino acids 60-98, amino acids103-141, amino acids 148-190, amino acids 195-234, or amino acids241-285 of SEQ ID NO: 18.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-234, or amino acids 241-285 of SEQ ID NO: 18. In other aspects ofthis embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-234, or amino acids241-285 of SEQ ID NO: 18. In yet other aspects of this embodiment, abinding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18.In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-234, or amino acids 241-285 of SEQ ID NO: 18. In still other aspectsof this embodiment, a binding domain comprising a BoNT/C1 HA-33β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-234, or amino acids241-285 of SEQ ID NO: 18. In other aspects of this embodiment, a bindingdomain comprising a BoNT/C1 HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 18.

In another embodiment, a binding domain comprising a BoNT/C1 HA-33β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domainof a BoNT/C1 HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of aBoNT/C1 HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/C1HA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/C1 HA-33of SEQ ID NO: 18. In another aspect of this embodiment, a binding domaincomprising a BoNT/C1 HA-33 β-trefoil domain comprises amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQID NO: 18.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, atleast 75% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, atleast 80% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, atleast 85% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, atleast 90% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18 orat least 95% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18. Inyet other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, at most75% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, at most80% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, at most85% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18, at most90% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18 or at most95% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 99-102, amino acids 191-194,or amino acids 235-240 of SEQ ID NO: 18. In other aspects of thisembodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQID NO: 18. In other aspects of this embodiment, a non-contiguous aminoacid substitution of any amino acid from amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18 canbe replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 235-240 of SEQ ID NO: 18 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a BoNT/C1 HA-33 comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18. In otheraspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 55-59, amino acids 99-102, amino acids 191-194, or amino acids235-240 of SEQ ID NO: 18. In still other aspects of this embodiment, abinding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid additions relative to amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18. In otheraspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 55-59, amino acids 99-102, amino acids 191-194, or amino acids235-240 of SEQ ID NO: 18.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 99-102, amino acids 191-194,or amino acids 235-240 of SEQ ID NO: 18. In other aspects of thisembodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO:18. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 18 can bereplaced with glycine. In other aspects of this embodiment, contiguousamino acid substitutions of hydrophobic amino acids from amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240of SEQ ID NO: 18 can be replaced with phenylalanine. In yet otheraspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid deletions relative to amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240of SEQ ID NO: 18. In other aspects of this embodiment, a binding domaincomprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 55-59, amino acids 99-102, amino acids191-194, or amino acids 235-240 of SEQ ID NO: 18. In still other aspectsof this embodiment, a binding domain comprising a BoNT/C1 HA-33β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240of SEQ ID NO: 18. In other aspects of this embodiment, a binding domaincomprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 55-59, amino acids 99-102, amino acids191-194, or amino acids 235-240 of SEQ ID NO: 18.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/C1 HA-33 of SEQ ID NO: 19. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/C1HA-33 comprises amino acids 10-141 or amino acids 148-286 of SEQ ID NO:19. In another aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/C1 HA-33 comprises a 1α-fold motifof a β-trefoil domain of a BoNT/C1 HA-33, a 1β-fold motif of a β-trefoildomain of a BoNT/C1 HA-33, a 1γ-fold motif of a β-trefoil domain of aBoNT/C1 HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/C1 HA-33,a 2α-fold motif of a β-trefoil domain of a BoNT/C1 HA-33, or a 2γ-foldmotif of a β-trefoil domain of a BoNT/C1 HA-33 of SEQ ID NO: 19. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/C1 HA-33 comprises amino acids10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190,amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 10-54, amino acids 60-98,amino acids 103-141, amino acids 148-190, amino acids 195-235, or aminoacids 242-286 of SEQ ID NO: 19, at least 75% amino acid identity withamino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19,at least 80% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 9, at least 85% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:19, at least 90% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, oramino acids 242-286 of SEQ ID NO: 19 or at least 95% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:19. In yet other aspects of this embodiment, a binding domain comprisinga BoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, oramino acids 242-286 of SEQ ID NO: 19, at most 75% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:19, at most 80% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, oramino acids 242-286 of SEQ ID NO: 19, at most 85% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:19, at most 90% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, oramino acids 242-286 of SEQ ID NO: 19 or at most 95% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:19.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-54,amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 19. In other aspects ofthis embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 19. In yet other aspects of this embodiment, abinding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-98, amino acids 103-141,amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQID NO: 19. In other aspects of this embodiment, a binding domaincomprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19.In still other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 19. In other aspects ofthis embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 10-54, amino acids 60-98, amino acids103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 19.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 19. In other aspects ofthis embodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 19. In yet other aspects of this embodiment, abinding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19.In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 19. In still other aspectsof this embodiment, a binding domain comprising a BoNT/C1 HA-33β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 19. In other aspects of this embodiment, a bindingdomain comprising a BoNT/C1 HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 19.

In another embodiment, a binding domain comprising a BoNT/C1 HA-33β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domainof a BoNT/C1 HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of aBoNT/C1 HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/C1HA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/C1 HA-33of SEQ ID NO: 19. In another aspect of this embodiment, a binding domaincomprising a BoNT/C1 HA-33 β-trefoil domain comprises amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQID NO: 19.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, atleast 75% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, atleast 80% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, atleast 85% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, atleast 90% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19 orat least 95% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19. Inyet other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, at most75% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, at most80% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, at most85% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19, at most90% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19 or at most95% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 99-102, amino acids 191-194,or amino acids 236-241 of SEQ ID NO: 19. In other aspects of thisembodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQID NO: 19. In other aspects of this embodiment, a non-contiguous aminoacid substitution of any amino acid from amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19 canbe replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 19 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a BoNT/C1 HA-33 comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19. In otheraspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 55-59, amino acids 99-102, amino acids 191-194, or amino acids236-241 of SEQ ID NO: 19. In still other aspects of this embodiment, abinding domain comprising a BoNT/C1 HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid additions relative to amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19. In otheraspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 55-59, amino acids 99-102, amino acids 191-194, or amino acids236-241 of SEQ ID NO: 19.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 99-102, amino acids 191-194,or amino acids 236-241 of SEQ ID NO: 19. In other aspects of thisembodiment, a binding domain comprising a BoNT/C1 HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO:19. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 19 can bereplaced with glycine. In other aspects of this embodiment, contiguousamino acid substitutions of hydrophobic amino acids from amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241of SEQ ID NO: 19 can be replaced with phenylalanine. In yet otheraspects of this embodiment, a binding domain comprising a BoNT/C1 HA-33β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid deletions relative to amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241of SEQ ID NO: 19. In other aspects of this embodiment, a binding domaincomprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 55-59, amino acids 99-102, amino acids191-194, or amino acids 236-241 of SEQ ID NO: 19. In still other aspectsof this embodiment, a binding domain comprising a BoNT/C1 HA-33β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241of SEQ ID NO: 19. In other aspects of this embodiment, a binding domaincomprising a BoNT/C1 HA-33 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 55-59, amino acids 99-102, amino acids191-194, or amino acids 236-241 of SEQ ID NO: 19.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/D HA-33 of SEQ ID NO: 20. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/D HA-33comprises amino acids 10-141 or amino acids 148-286 of SEQ ID NO: 20. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/D HA-33 comprises a 1α-fold motifof a β-trefoil domain of a BoNT/D HA-33, a 1β-fold motif of a β-trefoildomain of a BoNT/D HA-33, a 1γ-fold motif of a β-trefoil domain of aBoNT/D HA-33, a 2α-fold motif of a β-trefoil domain of a BoNT/D HA-33, a2α-fold motif of a β-trefoil domain of a BoNT/D HA-33, or a 2γ-foldmotif of a β-trefoil domain of a BoNT/D HA-33 of SEQ ID NO: 20. Inanother aspect of this embodiment, a binding domain comprising aβ-trefoil domain derived from a BoNT/D HA-33 comprises amino acids10-54, amino acids 60-98, amino acids 103-141, amino acids 148-190,amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20.

In other aspects of this embodiment, a binding domain comprising aBoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 10-54, amino acids 60-98,amino acids 103-141, amino acids 148-190, amino acids 195-235, or aminoacids 242-286 of SEQ ID NO: 20, at least 75% amino acid identity withamino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20,at least 80% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 9, at least 85% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:20, at least 90% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, oramino acids 242-286 of SEQ ID NO: 20 or at least 95% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:20. In yet other aspects of this embodiment, a binding domain comprisinga BoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 10-54, amino acids 60-98,amino acids 103-141, amino acids 148-190, amino acids 195-235, or aminoacids 242-286 of SEQ ID NO: 20, at most 75% amino acid identity withamino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20,at most 80% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, oramino acids 242-286 of SEQ ID NO: 20, at most 85% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:20, at most 90% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, oramino acids 242-286 of SEQ ID NO: 20 or at most 95% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:20.

In other aspects of this embodiment, a binding domain comprising aBoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-54,amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 20. In other aspects ofthis embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 20. In yet other aspects of this embodiment, abinding domain comprising a BoNT/D HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-98, amino acids 103-141,amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQID NO: 20. In other aspects of this embodiment, a binding domaincomprising a BoNT/D HA-33 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20.In still other aspects of this embodiment, a binding domain comprising aBoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 20. In other aspects ofthis embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 10-54, amino acids 60-98, amino acids103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 20.

In other aspects of this embodiment, a binding domain comprising aBoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 20. In other aspects ofthis embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 20. In yet other aspects of this embodiment, abinding domain comprising a BoNT/D HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20.In other aspects of this embodiment, a binding domain comprising aBoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 20. In still other aspectsof this embodiment, a binding domain comprising a BoNT/D HA-33 β-trefoildomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 10-54, amino acids 60-98, amino acids103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 20. In other aspects of this embodiment, a bindingdomain comprising a BoNT/D HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 20.

In another embodiment, a binding domain comprising a BoNT/D HA-33β-trefoil domain comprises a 1β4/β5 hairpin turn of a β-trefoil domainof a BoNT/D HA-33, a 1β8/β9 hairpin turn of a β-trefoil domain of aBoNT/D HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a BoNT/DHA-33 or a 2β8/β9 hairpin turn of a β-trefoil domain of a BoNT/D HA-33of SEQ ID NO: 20. In another aspect of this embodiment, a binding domaincomprising a BoNT/D HA-33 β-trefoil domain comprises amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQID NO: 20.

In other aspects of this embodiment, a binding domain comprising aBoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, atleast 75% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, atleast 80% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, atleast 85% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, atleast 90% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20 orat least 95% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20. Inyet other aspects of this embodiment, a binding domain comprising aBoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, at most75% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, at most80% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, at most85% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20, at most90% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20 or at most95% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20.

In other aspects of this embodiment, a binding domain comprising aBoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 99-102, amino acids 191-194,or amino acids 236-241 of SEQ ID NO: 20. In other aspects of thisembodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQID NO: 20. In other aspects of this embodiment, a non-contiguous aminoacid substitution of any amino acid from amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20 canbe replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 20 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a BoNT/D HA-33 comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20. In otheraspects of this embodiment, a binding domain comprising a BoNT/D HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 55-59, amino acids 99-102, amino acids 191-194, or amino acids236-241 of SEQ ID NO: 20. In still other aspects of this embodiment, abinding domain comprising a BoNT/D HA-33 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid additions relative to amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20. In otheraspects of this embodiment, a binding domain comprising a BoNT/D HA-33β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 55-59, amino acids 99-102, amino acids 191-194, or amino acids236-241 of SEQ ID NO: 20.

In other aspects of this embodiment, a binding domain comprising aBoNT/D HA-33 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 55-59, amino acids 99-102, amino acids 191-194,or amino acids 236-241 of SEQ ID NO: 20. In other aspects of thisembodiment, a binding domain comprising a BoNT/D HA-33 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO:20. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20 can bereplaced with glycine. In other aspects of this embodiment, contiguousamino acid substitutions of hydrophobic amino acids from amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241of SEQ ID NO: 20 can be replaced with phenylalanine. In yet otheraspects of this embodiment, a binding domain comprising a BoNT/DHA-33β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four contiguous amino acid deletions relative to aminoacids 55-59, amino acids 99-102, amino acids 191-194, or amino acids236-241 of SEQ ID NO: 20. In other aspects of this embodiment, a bindingdomain comprising a BoNT/D HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid deletions relative to amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20. In stillother aspects of this embodiment, a binding domain comprising a BoNT/DHA-33 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 55-59, amino acids 99-102, amino acids 191-194, or aminoacids 236-241 of SEQ ID NO: 20. In other aspects of this embodiment, abinding domain comprising a BoNT/D HA-33 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid additions relative to amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 20.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a Clostridial HA-17. In an aspect of this embodiment, aβ-trefoil domain derived from a Clostridial HA-17 comprises, e.g., aβ-trefoil domain derived from a BoNT/A HA-17, a β-trefoil domain derivedfrom a BoNT/B HA-17, a β-trefoil domain derived from a BoNT/C1 HA-17 ora β-trefoil domain derived from a BoNT/D HA-17. In another aspect ofthis embodiment, a β-trefoil domain derived from a Clostridial HA-17comprises an α-fold motif of a β-trefoil domain of a BoNT/A HA-17, aβ-fold motif of a β-trefoil domain of a BoNT/A HA-17, or a γ-fold motifof a β-trefoil domain of a BoNT/A HA-17. In another aspect of thisembodiment, a β-trefoil domain derived from a Clostridial HA-17comprises an α-fold motif of a β-trefoil domain of a BoNT/B HA-17, aβ-fold motif of a β-trefoil domain of a BoNT/B HA-17, or a γ-fold motifof a β-trefoil domain of a BoNT/B HA-17. In another aspect of thisembodiment, a β-trefoil domain derived from a Clostridial HA-17comprises an α-fold motif of a β-trefoil domain of a BoNT/C1 HA-17, aβ-fold motif of a β-trefoil domain of a BoNT/C1 HA-17, or a γ-fold motifof a β-trefoil domain of a BoNT/C1 HA-17. In another aspect of thisembodiment, a β-trefoil domain derived from a Clostridial HA-17comprises an α-fold motif of a β-trefoil domain of a BoNT/D HA-17, aβ-fold motif of a β-trefoil domain of a BoNT/D HA-17, or a γ-fold motifof a β-trefoil domain of a BoNT/D HA-17.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/A HA-17 of SEQ ID NO: 21. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/A HA-17comprises amino acids 9-146 of SEQ ID NO: 21. In another aspect of thisembodiment, a binding domain comprising a β-trefoil domain derived froma BoNT/A HA-17 comprises a α-fold motif of a β-trefoil domain of aBoNT/A HA-17, a β-fold motif of a β-trefoil domain of a BoNT/A HA-17 ora γ-fold motif of a β-trefoil domain of a BoNT/A HA-17 of SEQ ID NO: 21.In another aspect of this embodiment, a β-trefoil domain derived from aBoNT/A HA-17 comprises amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 21.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 9-50, amino acids 55-91,or amino acids 95-146 of SEQ ID NO: 21, at least 75% amino acid identitywith amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQID NO: 21, at least 80% amino acid identity with amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 21, at least 85% aminoacid identity with amino acids 9-50, amino acids 55-91, or amino acids95-146 of SEQ ID NO: 21, at least 90% amino acid identity with aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21 orat least 95% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 21. In yet other aspects ofthis embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoildomain comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 21, at most 75% amino acid identity with amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21, at most 80%amino acid identity with amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 21, at most 85% amino acid identity withamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:21, at most 90% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 21 or at most 95% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 21.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In otheraspects of this embodiment, a binding domain comprising a BoNT/A HA-17β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 21. In yet other aspects ofthis embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoildomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 21. In other aspects of this embodiment, abinding domain comprising a BoNT/A HA-17 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 21. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/A HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 21. In other aspects of this embodiment, a binding domaincomprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In other aspects ofthis embodiment, a binding domain comprising a BoNT/A HA-17 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 21. In yet other aspects of this embodiment,a binding domain comprising a BoNT/A HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:21. In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In still otheraspects of this embodiment, a binding domain comprising a BoNT/A HA-17β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 21. In other aspects of this embodiment, abinding domain comprising a BoNT/A HA-17 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:21.

In another embodiment, a binding domain comprising a BoNT/A HA-17β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain ofa BoNT/A HA-17 or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/AHA-17 of SEQ ID NO: 21. In another aspect of this embodiment, a bindingdomain comprising a BoNT/A HA-17 β-trefoil domain comprises amino acids51-54 or amino acids 92-94 of SEQ ID NO: 21.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 51-54 or amino acids92-94 of SEQ ID NO: 21, at least 75% amino acid identity with aminoacids 51-54 or amino acids 92-94 of SEQ ID NO: 21, at least 80% aminoacid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO:21, at least 85% amino acid identity with amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 21, at least 90% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21 or at least 95%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 21. In yet other aspects of this embodiment, a binding domaincomprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 21, at most 75% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21, at most 80%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 21, at most 85% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 21, at most 90% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21 or at most 95%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 21.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. Inother aspects of this embodiment, a binding domain comprising a BoNT/AHA-17 β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three or four non-contiguous amino acid substitutions relativeto amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany amino acid from amino acids 51-54 or amino acids 92-94 of SEQ ID NO:21 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21 can bereplaced with phenylalanine. In yet other aspects of this embodiment, abinding domain comprising a BoNT/A HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 51-54 or amino acids 92-94of SEQ ID NO: 21. In other aspects of this embodiment, a binding domaincomprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino aciddeletions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 21. In still other aspects of this embodiment, a binding domaincomprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at most one, two, three or four non-contiguous amino acidadditions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 21. In other aspects of this embodiment, a binding domain comprisinga BoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four non-contiguous amino acid additionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21.

In other aspects of this embodiment, a binding domain comprising aBoNT/A HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. Inother aspects of this embodiment, a binding domain comprising a BoNT/AHA-17 β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three or four contiguous amino acid substitutions relative toamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. In otheraspects of this embodiment, contiguous amino acid substitutions of aminoacids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21 canbe replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21 can be replacedwith phenylalanine. In yet other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a BoNT/A HA-17comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 21. In other aspects of this embodiment, abinding domain comprising a BoNT/A HA-17 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid deletions relative to amino acids 51-54 or amino acids 92-94of SEQ ID NO: 21. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/A HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four contiguousamino acid additions relative to amino acids 51-54 or amino acids 92-94of SEQ ID NO: 21. In other aspects of this embodiment, a binding domaincomprising a BoNT/A HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 21.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/B HA-17 of SEQ ID NO: 22. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/B HA-17comprises amino acids 9-146 of SEQ ID NO: 22. In another aspect of thisembodiment, a binding domain comprising a β-trefoil domain derived froma BoNT/B HA-17 comprises a α-fold motif of a β-trefoil domain of aBoNT/B HA-17, a β-fold motif of a β-trefoil domain of a BoNT/B HA-17 ora γ-fold motif of a β-trefoil domain of a BoNT/B HA-17 of SEQ ID NO: 22.In another aspect of this embodiment, a β-trefoil domain derived from aBoNT/B HA-17 comprises amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 22.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 9-50, amino acids 55-91,or amino acids 95-146 of SEQ ID NO: 22, at least 75% amino acid identitywith amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQID NO: 22, at least 80% amino acid identity with amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 22, at least 85% aminoacid identity with amino acids 9-50, amino acids 55-91, or amino acids95-146 of SEQ ID NO: 22, at least 90% amino acid identity with aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22 orat least 95% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 22. In yet other aspects ofthis embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoildomain comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 22, at most 75% amino acid identity with amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22, at most 80%amino acid identity with amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 22, at most 85% amino acid identity withamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:22, at most 90% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 22 or at most 95% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 22.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In otheraspects of this embodiment, a binding domain comprising a BoNT/B HA-17β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 22. In yet other aspects ofthis embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoildomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 22. In other aspects of this embodiment, abinding domain comprising a BoNT/B HA-17 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 22. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/B HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 22. In other aspects of this embodiment, a binding domaincomprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In other aspects ofthis embodiment, a binding domain comprising a BoNT/B HA-17 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 22. In yet other aspects of this embodiment,a binding domain comprising a BoNT/B HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:22. In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In still otheraspects of this embodiment, a binding domain comprising a BoNT/B HA-17β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 22. In other aspects of this embodiment, abinding domain comprising a BoNT/B HA-17 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:22.

In another embodiment, a binding domain comprising a BoNT/B HA-17β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain ofa BoNT/B HA-17 or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/BHA-17 of SEQ ID NO: 22. In another aspect of this embodiment, a bindingdomain comprising a BoNT/B HA-17 β-trefoil domain comprises amino acids51-54 or amino acids 92-94 of SEQ ID NO: 22.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 51-54 or amino acids92-94 of SEQ ID NO: 22, at least 75% amino acid identity with aminoacids 51-54 or amino acids 92-94 of SEQ ID NO: 22, at least 80% aminoacid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO:22, at least 85% amino acid identity with amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 22, at least 90% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22 or at least 95%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 22. In yet other aspects of this embodiment, a binding domaincomprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 22, at most 75% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22, at most 80%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 22, at most 85% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 22, at most 90% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22 or at most 95%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 22.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. Inother aspects of this embodiment, a binding domain comprising a BoNT/BHA-17 β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three or four non-contiguous amino acid substitutions relativeto amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany amino acid from amino acids 51-54 or amino acids 92-94 of SEQ ID NO:22 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22 can bereplaced with phenylalanine. In yet other aspects of this embodiment, abinding domain comprising a BoNT/B HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 51-54 or amino acids 92-94of SEQ ID NO: 22. In other aspects of this embodiment, a binding domaincomprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino aciddeletions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 22. In still other aspects of this embodiment, a binding domaincomprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at most one, two, three or four non-contiguous amino acidadditions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 22. In other aspects of this embodiment, a binding domain comprisinga BoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four non-contiguous amino acid additionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22.

In other aspects of this embodiment, a binding domain comprising aBoNT/B HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. Inother aspects of this embodiment, a binding domain comprising a BoNT/BHA-17 β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three or four contiguous amino acid substitutions relative toamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. In otheraspects of this embodiment, contiguous amino acid substitutions of aminoacids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22 canbe replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22 can be replacedwith phenylalanine. In yet other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a BoNT/B HA-17comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 22. In other aspects of this embodiment, abinding domain comprising a BoNT/B HA-17 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid deletions relative to amino acids 51-54 or amino acids 92-94of SEQ ID NO: 22. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/B HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four contiguousamino acid additions relative to amino acids 51-54 or amino acids 92-94of SEQ ID NO: 22. In other aspects of this embodiment, a binding domaincomprising a BoNT/B HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 22.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/C1 HA-17 of SEQ ID NO: 23. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/C1HA-17 comprises amino acids 9-146 of SEQ ID NO: 23. In another aspect ofthis embodiment, a binding domain comprising a β-trefoil domain derivedfrom a BoNT/C1 HA-17 comprises a α-fold motif of a β-trefoil domain of aBoNT/C1 HA-17, a β-fold motif of a β-trefoil domain of a BoNT/C1 HA-17or a γ-fold motif of a β-trefoil domain of a BoNT/C1 HA-17 of SEQ ID NO:23. In another aspect of this embodiment, a β-trefoil domain derivedfrom a BoNT/C1 HA-17 comprises amino acids 9-50, amino acids 55-91, oramino acids 95-146 of SEQ ID NO: 23.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 9-50, amino acids 55-91,or amino acids 95-146 of SEQ ID NO: 23, at least 75% amino acid identitywith amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQID NO: 23, at least 80% amino acid identity with amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 23, at least 85% aminoacid identity with amino acids 9-50, amino acids 55-91, or amino acids95-146 of SEQ ID NO: 23, at least 90% amino acid identity with aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23 orat least 95% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 23. In yet other aspects ofthis embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoildomain comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 23, at most 75% amino acid identity with amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23, at most 80%amino acid identity with amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 23, at most 85% amino acid identity withamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:23, at most 90% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 23 or at most 95% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 23.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23. In otheraspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 23. In yet other aspects ofthis embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoildomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 23. In other aspects of this embodiment, abinding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 23. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/C1 HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 23. In other aspects of this embodiment, a binding domaincomprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 23.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 23. In other aspects ofthis embodiment, a binding domain comprising a BoNT/C1 HA-17 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 23. In yet other aspects of this embodiment,a binding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:23. In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 23. In still otheraspects of this embodiment, a binding domain comprising a BoNT/C1 HA-17β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 23. In other aspects of this embodiment, abinding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:23.

In another embodiment, a binding domain comprising a BoNT/C1 HA-17β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain ofa BoNT/C1 HA-17 or a β8/β9 hairpin turn of a β-trefoil domain of aBoNT/C1 HA-17 of SEQ ID NO: 23. In another aspect of this embodiment, abinding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprisesamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 51-54 or amino acids92-94 of SEQ ID NO: 23, at least 75% amino acid identity with aminoacids 51-54 or amino acids 92-94 of SEQ ID NO: 23, at least 80% aminoacid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO:23, at least 85% amino acid identity with amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 23, at least 90% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23 or at least 95%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 23. In yet other aspects of this embodiment, a binding domaincomprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 23, at most 75% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23, at most 80%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 23, at most 85% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 23, at most 90% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23 or at most 95%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 23.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23. Inother aspects of this embodiment, a binding domain comprising a BoNT/C1HA-17 β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three or four non-contiguous amino acid substitutions relativeto amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany amino acid from amino acids 51-54 or amino acids 92-94 of SEQ ID NO:23 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23 can bereplaced with phenylalanine. In yet other aspects of this embodiment, abinding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 51-54 or amino acids 92-94of SEQ ID NO: 23. In other aspects of this embodiment, a binding domaincomprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino aciddeletions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 23. In still other aspects of this embodiment, a binding domaincomprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at most one, two, three or four non-contiguous amino acidadditions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 23. In other aspects of this embodiment, a binding domain comprisinga BoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g.,at least one, two, three or four non-contiguous amino acid additionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23. Inother aspects of this embodiment, a binding domain comprising a BoNT/C1HA-17 β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three or four contiguous amino acid substitutions relative toamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23. In otheraspects of this embodiment, contiguous amino acid substitutions of aminoacids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23 canbe replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 23 can be replacedwith phenylalanine. In yet other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a BoNT/C1 HA-17comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 23. In other aspects of this embodiment, abinding domain comprising a BoNT/C1 HA-17 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid deletions relative to amino acids 51-54 or amino acids 92-94of SEQ ID NO: 23. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/C1 HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four contiguousamino acid additions relative to amino acids 51-54 or amino acids 92-94of SEQ ID NO: 23. In other aspects of this embodiment, a binding domaincomprising a BoNT/C1 HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 23.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/D HA-17 of SEQ ID NO: 24. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/D HA-17comprises amino acids 9-146 of SEQ ID NO: 24. In another aspect of thisembodiment, a binding domain comprising a β-trefoil domain derived froma BoNT/D HA-17 comprises a α-fold motif of a β-trefoil domain of aBoNT/D HA-17, a β-fold motif of a β-trefoil domain of a BoNT/D HA-17 ora γ-fold motif of a β-trefoil domain of a BoNT/D HA-17 of SEQ ID NO: 24.In another aspect of this embodiment, a β-trefoil domain derived from aBoNT/D HA-17 comprises amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 24.

In other aspects of this embodiment, a binding domain comprising aBoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 9-50, amino acids 55-91,or amino acids 95-146 of SEQ ID NO: 24, at least 75% amino acid identitywith amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQID NO: 24, at least 80% amino acid identity with amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 24, at least 85% aminoacid identity with amino acids 9-50, amino acids 55-91, or amino acids95-146 of SEQ ID NO: 24, at least 90% amino acid identity with aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24 orat least 95% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 24. In yet other aspects ofthis embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoildomain comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 24, at most 75% amino acid identity with amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24, at most 80%amino acid identity with amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 24, at most 85% amino acid identity withamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:24, at most 90% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 24 or at most 95% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 24.

In other aspects of this embodiment, a binding domain comprising aBoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24. In otheraspects of this embodiment, a binding domain comprising a BoNT/D HA-17β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 24. In yet other aspects ofthis embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoildomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 24. In other aspects of this embodiment, abinding domain comprising a BoNT/D HA-17 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 24. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/D HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 24. In other aspects of this embodiment, a binding domaincomprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 24.

In other aspects of this embodiment, a binding domain comprising aBoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 24. In other aspects ofthis embodiment, a binding domain comprising a BoNT/D HA-17 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 24. In yet other aspects of this embodiment,a binding domain comprising a BoNT/D HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:24. In other aspects of this embodiment, a binding domain comprising aBoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 24. In still otheraspects of this embodiment, a binding domain comprising a BoNT/D HA-17β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 24. In other aspects of this embodiment, abinding domain comprising a BoNT/D HA-17 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:24.

In another embodiment, a binding domain comprising a BoNT/D HA-17β-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain ofa BoNT/D HA-17 or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/DHA-17 of SEQ ID NO: 24. In another aspect of this embodiment, a bindingdomain comprising a BoNT/D HA-17 β-trefoil domain comprises amino acids51-54 or amino acids 92-94 of SEQ ID NO: 24.

In other aspects of this embodiment, a binding domain comprising aBoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 51-54 or amino acids92-94 of SEQ ID NO: 24, at least 75% amino acid identity with aminoacids 51-54 or amino acids 92-94 of SEQ ID NO: 24, at least 80% aminoacid identity with amino acids 51-54 or amino acids 92-94 of SEQ ID NO:24, at least 85% amino acid identity with amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 24, at least 90% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24 or at least 95%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 24. In yet other aspects of this embodiment, a binding domaincomprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 24, at most 75% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24, at most 80%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 24, at most 85% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 24, at most 90% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24 or at most 95%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 24.

In other aspects of this embodiment, a binding domain comprising aBoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24. Inother aspects of this embodiment, a binding domain comprising a BoNT/DHA-17 β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three or four non-contiguous amino acid substitutions relativeto amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany amino acid from amino acids 51-54 or amino acids 92-94 of SEQ ID NO:24 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24 can bereplaced with phenylalanine. In yet other aspects of this embodiment, abinding domain comprising a BoNT/D HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 51-54 or amino acids 92-94of SEQ ID NO: 24. In other aspects of this embodiment, a binding domaincomprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino aciddeletions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 24. In still other aspects of this embodiment, a binding domaincomprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at most one, two, three or four non-contiguous amino acidadditions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 24. In other aspects of this embodiment, a binding domain comprisinga BoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four non-contiguous amino acid additionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24.

In other aspects of this embodiment, a binding domain comprising aBoNT/D HA-17 β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24. Inother aspects of this embodiment, a binding domain comprising a BoNT/DHA-17 β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three or four contiguous amino acid substitutions relative toamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24. In otheraspects of this embodiment, contiguous amino acid substitutions of aminoacids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24 canbe replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 24 can be replacedwith phenylalanine. In yet other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a BoNT/D HA-17comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 24. In other aspects of this embodiment, abinding domain comprising a BoNT/D HA-17 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid deletions relative to amino acids 51-54 or amino acids 92-94of SEQ ID NO: 24. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/D HA-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four contiguousamino acid additions relative to amino acids 51-54 or amino acids 92-94of SEQ ID NO: 24. In other aspects of this embodiment, a binding domaincomprising a BoNT/D HA-17 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 24.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a Clostridial NTNH. In an aspect of this embodiment, abinding domain comprising a β-trefoil domain derived from a ClostridialNTNH comprises, e.g., a β-trefoil domain derived from a BoNT/A NTNH, aβ-trefoil domain derived from a BoNT/B NTNH, a β-trefoil domain derivedfrom a BoNT/C1 NTNH, a β-trefoil domain derived from a BoNT/D NTNH, aβ-trefoil domain derived from a BoNT/E NTNH, a β-trefoil domain derivedfrom a BoNT/F NTNH, or a β-trefoil domain derived from a BoNT/G NTNH. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial NTNH comprises an α-fold motif of a β-trefoil domain of aBoNT/A NTNH, β-fold motif of β-trefoil domain of a BoNT/A NTNH, or aγ-fold motif of a β-trefoil domain of a BoNT/A NTNH. In another aspectof this embodiment, a β-trefoil domain derived from a Clostridial NTNHcomprises an α-fold motif of a β-trefoil domain of a BoNT/B NTNH, aβ-fold motif of a β-trefoil domain of a BoNT/B NTNH, or a γ-fold motifof a β-trefoil domain of a BoNT/B NTNH. In another aspect of thisembodiment, a β-trefoil domain derived from a Clostridial NTNH comprisesan α-fold motif of a β-trefoil domain of a BoNT/C1 NTNH, a β-fold motifof a β-trefoil domain of a BoNT/C1 NTNH, or a γ-fold motif of aβ-trefoil domain of a BoNT/C1 NTNH. In another aspect of thisembodiment, a β-trefoil domain derived from a Clostridial NTNH comprisesan β-fold motif of a β-trefoil domain of a BoNT/D NTNH, a β-fold motifof a β-trefoil domain of a BoNT/D NTNH, or a γ-fold motif of a β-trefoildomain of a BoNT/D NTNH. In another aspect of this embodiment, aβ-trefoil domain derived from a Clostridial NTNH comprises an β-foldmotif of a β-trefoil domain of a BoNT/E NTNH, a β-fold motif of aβ-trefoil domain of a BoNT/E NTNH, or a γ-fold motif of a β-trefoildomain of a BoNT/E NTNH. In another aspect of this embodiment, aβ-trefoil domain derived from a Clostridial NTNH comprises an α-foldmotif of a β-trefoil domain of a BoNT/F NTNH, a β-fold motif of aβ-trefoil domain of a BoNT/F NTNH, or a γ-fold motif of a β-trefoildomain of a BoNT/F NTNH. In another aspect of this embodiment, aβ-trefoil domain derived from a Clostridial NTNH comprises an α-foldmotif of a β-trefoil domain of a BoNT/G NTNH, a β-fold motif of aβ-trefoil domain of a BoNT/G NTNH, or a γ-fold motif of a β-trefoildomain of a BoNT/G NTNH.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/A NTNH of SEQ ID NO: 25. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/A NTNHcomprises amino acids 1050-1194 of SEQ ID NO: 25. In another aspect ofthis embodiment, a binding domain comprising a β-trefoil domain derivedfrom a BoNT/A NTNH comprises a α-fold motif of a β-trefoil domain of aBoNT/A NTNH, a β-fold motif of a β-trefoil domain of a BoNT/A NTNH or aγ-fold motif of a β-trefoil domain of a BoNT/A NTNH of SEQ ID NO: 24. Inanother aspect of this embodiment, a β-trefoil domain derived from aBoNT/A NTNH comprises amino acids 1050-1097, amino acids 1111-1138, oramino acids 1149-1194 of SEQ ID NO: 25.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1050-1097, amino acids1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25, at least 75% aminoacid identity with amino acids 1050-1097, amino acids 1111-1138, oramino acids 1149-1194 of SEQ ID NO: 25, at least 80% amino acid identitywith amino acids 1050-1097, amino acids 1111-1138, or amino acids1149-1194 of SEQ ID NO: 25, at least 85% amino acid identity with aminoacids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQID NO: 25, at least 90% amino acid identity with amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25 or atleast 95% amino acid identity with amino acids 1050-1097, amino acids1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In yet otheraspects of this embodiment, a binding domain comprising a BoNT/A NTNHβ-trefoil domain comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1050-1097, amino acids 1111-1138, oramino acids 1149-1194 of SEQ ID NO: 25, at most 75% amino acid identitywith amino acids 1050-1097, amino acids 1111-1138, or amino acids1149-1194 of SEQ ID NO: 25, at most 80% amino acid identity with aminoacids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQID NO: 25, at most 85% amino acid identity with amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25, atmost 90% amino acid identity with amino acids 1050-1097, amino acids1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25 or at most 95%amino acid identity with amino acids 1050-1097, amino acids 1111-1138,or amino acids 1149-1194 of SEQ ID NO: 25.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO:25. In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO:25. In yet other aspects of this embodiment, a binding domain comprisinga BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. Inother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. Instill other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. Inother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. Inother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In yetother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 1050-1097, amino acids1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In other aspectsof this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 1050-1097, amino acids 1111-1138, oramino acids 1149-1194 of SEQ ID NO: 25. In still other aspects of thisembodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1050-1097, amino acids 1111-1138, or amino acids1149-1194 of SEQ ID NO: 25. In other aspects of this embodiment, abinding domain comprising a BoNT/A NTNH β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194of SEQ ID NO: 25.

In another embodiment, a binding domain comprising a BoNT/A NTNHβ-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain ofa BoNT/A NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/ANTNH of SEQ ID NO: 24. In another aspect of this embodiment, a bindingdomain comprising a BoNT/A NTNH β-trefoil domain comprises amino acids1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 25, at least 75% amino acid identity with aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, at least 80%amino acid identity with amino acids 1098-1110 or amino acids 1139-1148of SEQ ID NO: 25, at least 85% amino acid identity with amino acids1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, at least 90% aminoacid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQID NO: 25 or at least 95% amino acid identity with amino acids 1098-1110or amino acids 1139-1148 of SEQ ID NO: 25. In yet other aspects of thisembodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, atmost 75% amino acid identity with amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 25, at most 80% amino acid identity with aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, at most 85%amino acid identity with amino acids 1098-1110 or amino acids 1139-1148of SEQ ID NO: 25, at most 90% amino acid identity with amino acids1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25 or at most 95% aminoacid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQID NO: 25.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO:25. In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO:25. In other aspects of this embodiment, a non-contiguous amino acidsubstitution of any amino acid from amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 25 can be replaced with glycine. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany hydrophobic amino acid from amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 25 can be replaced with phenylalanine. In yetother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In otheraspects of this embodiment, a binding domain comprising a BoNT/A NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In stillother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid additions relative to aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In otheraspects of this embodiment, a binding domain comprising a BoNT/A NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO:25. In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO:25. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 25 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 25 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a BoNT/A NTNH comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1139-1148 ofSEQ ID NO: 25. In other aspects of this embodiment, a binding domaincomprising a BoNT/A NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1139-1148 ofSEQ ID NO: 25. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino acidadditions relative to amino acids 1098-1110 or amino acids 1139-1148 ofSEQ ID NO: 25. In other aspects of this embodiment, a binding domaincomprising a BoNT/A NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 1098-1110 or amino acids 1139-1148 ofSEQ ID NO: 25.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/A NTNH of SEQ ID NO: 26. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/A NTNHcomprises amino acids 1050-1199 of SEQ ID NO: 26. In another aspect ofthis embodiment, a binding domain comprising a β-trefoil domain derivedfrom a BoNT/A NTNH comprises a α-fold motif of a β-trefoil domain of aBoNT/A NTNH, a β-fold motif of a β-trefoil domain of a BoNT/A NTNH or aγ-fold motif of a β-trefoil domain of a BoNT/A NTNH of SEQ ID NO: 24. Inanother aspect of this embodiment, a β-trefoil domain derived from aBoNT/A NTNH comprises amino acids 1050-1097, amino acids 1111-1139, oramino acids 1149-1199 of SEQ ID NO: 26.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1050-1097, amino acids1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26, at least 75% aminoacid identity with amino acids 1050-1097, amino acids 1111-1139, oramino acids 1149-1199 of SEQ ID NO: 26, at least 80% amino acid identitywith amino acids 1050-1097, amino acids 1111-1139, or amino acids1149-1199 of SEQ ID NO: 26, at least 85% amino acid identity with aminoacids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQID NO: 26, at least 90% amino acid identity with amino acids 1050-1097,amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26 or atleast 95% amino acid identity with amino acids 1050-1097, amino acids1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. In yet otheraspects of this embodiment, a binding domain comprising a BoNT/A NTNHβ-trefoil domain comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1050-1097, amino acids 1111-1139, oramino acids 1149-1199 of SEQ ID NO: 26, at most 75% amino acid identitywith amino acids 1050-1097, amino acids 1111-1139, or amino acids1149-1199 of SEQ ID NO: 26, at most 80% amino acid identity with aminoacids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQID NO: 26, at most 85% amino acid identity with amino acids 1050-1097,amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26, atmost 90% amino acid identity with amino acids 1050-1097, amino acids1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26 or at most 95%amino acid identity with amino acids 1050-1097, amino acids 1111-1139,or amino acids 1149-1199 of SEQ ID NO: 26.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO:26. In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO:26. In yet other aspects of this embodiment, a binding domain comprisinga BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1050-1097,amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. Inother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1050-1097,amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. Instill other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1050-1097,amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. Inother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1050-1097,amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1050-1097,amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. Inother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1050-1097,amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. In yetother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 1050-1097, amino acids1111-1139, or amino acids 1149-1199 of SEQ ID NO: 26. In other aspectsof this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 1050-1097, amino acids 1111-1139, oramino acids 1149-1199 of SEQ ID NO: 26. In still other aspects of thisembodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1050-1097, amino acids 1111-1139, or amino acids1149-1199 of SEQ ID NO: 26. In other aspects of this embodiment, abinding domain comprising a BoNT/A NTNH β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199of SEQ ID NO: 26.

In another embodiment, a binding domain comprising a BoNT/A NTNHβ-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain ofa BoNT/A NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/ANTNH of SEQ ID NO: 24. In another aspect of this embodiment, a bindingdomain comprising a BoNT/A NTNH β-trefoil domain comprises amino acids1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1098-1110 or amino acids1140-1148 of SEQ ID NO: 26, at least 75% amino acid identity with aminoacids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26, at least 80%amino acid identity with amino acids 1098-1110 or amino acids 1140-1148of SEQ ID NO: 26, at least 85% amino acid identity with amino acids1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26, at least 90% aminoacid identity with amino acids 1098-1110 or amino acids 1140-1148 of SEQID NO: 26 or at least 95% amino acid identity with amino acids 1098-1110or amino acids 1140-1148 of SEQ ID NO: 26. In yet other aspects of thisembodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26, atmost 75% amino acid identity with amino acids 1098-1110 or amino acids1140-1148 of SEQ ID NO: 26, at most 80% amino acid identity with aminoacids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26, at most 85%amino acid identity with amino acids 1098-1110 or amino acids 1140-1148of SEQ ID NO: 26, at most 90% amino acid identity with amino acids1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26 or at most 95% aminoacid identity with amino acids 1098-1110 or amino acids 1140-1148 of SEQID NO: 26.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO:26. In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO:26. In other aspects of this embodiment, a non-contiguous amino acidsubstitution of any amino acid from amino acids 1098-1110 or amino acids1140-1148 of SEQ ID NO: 26 can be replaced with glycine. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany hydrophobic amino acid from amino acids 1098-1110 or amino acids1140-1148 of SEQ ID NO: 26 can be replaced with phenylalanine. In yetother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26. In otheraspects of this embodiment, a binding domain comprising a BoNT/A NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26. In stillother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid additions relative to aminoacids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26. In otheraspects of this embodiment, a binding domain comprising a BoNT/A NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 26.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO:26. In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO:26. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 1098-1110 or amino acids1140-1148 of SEQ ID NO: 26 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1098-1110 or amino acids1140-1148 of SEQ ID NO: 26 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a BoNT/A NTNH comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1140-1148 ofSEQ ID NO: 26. In other aspects of this embodiment, a binding domaincomprising a BoNT/A NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1140-1148 ofSEQ ID NO: 26. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino acidadditions relative to amino acids 1098-1110 or amino acids 1140-1148 ofSEQ ID NO: 26. In other aspects of this embodiment, a binding domaincomprising a BoNT/A NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 1098-1110 or amino acids 1140-1148 ofSEQ ID NO: 26.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/A NTNH of SEQ ID NO: 27. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/A NTNHcomprises amino acids 1050-1194 of SEQ ID NO: 27. In another aspect ofthis embodiment, a binding domain comprising a β-trefoil domain derivedfrom a BoNT/A NTNH comprises a α-fold motif of a β-trefoil domain of aBoNT/A NTNH, a β-fold motif of a β-trefoil domain of a BoNT/A NTNH or aγ-fold motif of a β-trefoil domain of a BoNT/A NTNH of SEQ ID NO: 24. Inanother aspect of this embodiment, a β-trefoil domain derived from aBoNT/A NTNH comprises amino acids 1050-1097, amino acids 1111-1138, oramino acids 1149-1194 of SEQ ID NO: 27.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1050-1097, amino acids1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27, at least 75% aminoacid identity with amino acids 1050-1097, amino acids 1111-1138, oramino acids 1149-1194 of SEQ ID NO: 27, at least 80% amino acid identitywith amino acids 1050-1097, amino acids 1111-1138, or amino acids1149-1194 of SEQ ID NO: 27, at least 85% amino acid identity with aminoacids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQID NO: 27, at least 90% amino acid identity with amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27 or atleast 95% amino acid identity with amino acids 1050-1097, amino acids1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. In yet otheraspects of this embodiment, a binding domain comprising a BoNT/A NTNHβ-trefoil domain comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1050-1097, amino acids 1111-1138, oramino acids 1149-1194 of SEQ ID NO: 27, at most 75% amino acid identitywith amino acids 1050-1097, amino acids 1111-1138, or amino acids1149-1194 of SEQ ID NO: 27, at most 80% amino acid identity with aminoacids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQID NO: 27, at most 85% amino acid identity with amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27, atmost 90% amino acid identity with amino acids 1050-1097, amino acids1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27 or at most 95%amino acid identity with amino acids 1050-1097, amino acids 1111-1138,or amino acids 1149-1194 of SEQ ID NO: 27.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO:27. In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO:27. In yet other aspects of this embodiment, a binding domain comprisinga BoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. Inother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. Instill other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. Inother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. Inother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. In yetother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 1050-1097, amino acids1111-1138, or amino acids 1149-1194 of SEQ ID NO: 27. In other aspectsof this embodiment, a binding domain comprising a BoNT/A NTNH β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 1050-1097, amino acids 1111-1138, oramino acids 1149-1194 of SEQ ID NO: 27. In still other aspects of thisembodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1050-1097, amino acids 1111-1138, or amino acids1149-1194 of SEQ ID NO: 27. In other aspects of this embodiment, abinding domain comprising a BoNT/A NTNH β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194of SEQ ID NO: 27.

In another embodiment, a binding domain comprising a BoNT/A NTNHβ-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain ofa BoNT/A NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/ANTNH of SEQ ID NO: 24. In another aspect of this embodiment, a bindingdomain comprising a BoNT/A NTNH β-trefoil domain comprises amino acids1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 27, at least 75% amino acid identity with aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27, at least 80%amino acid identity with amino acids 1098-1110 or amino acids 1139-1148of SEQ ID NO: 27, at least 85% amino acid identity with amino acids1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27, at least 90% aminoacid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQID NO: 27 or at least 95% amino acid identity with amino acids 1098-1110or amino acids 1139-1148 of SEQ ID NO: 27. In yet other aspects of thisembodiment, a binding domain comprising a BoNT/A NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27, atmost 75% amino acid identity with amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 27, at most 80% amino acid identity with aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27, at most 85%amino acid identity with amino acids 1098-1110 or amino acids 1139-1148of SEQ ID NO: 27, at most 90% amino acid identity with amino acids1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27 or at most 95% aminoacid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQID NO: 27.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO:27. In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO:27. In other aspects of this embodiment, a non-contiguous amino acidsubstitution of any amino acid from amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 27 can be replaced with glycine. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany hydrophobic amino acid from amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 27 can be replaced with phenylalanine. In yetother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27. In otheraspects of this embodiment, a binding domain comprising a BoNT/A NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27. In stillother aspects of this embodiment, a binding domain comprising a BoNT/ANTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid additions relative to aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27. In otheraspects of this embodiment, a binding domain comprising a BoNT/A NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 27.

In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO:27. In other aspects of this embodiment, a binding domain comprising aBoNT/A NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO:27. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 27 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 27 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a BoNT/A NTNH comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1139-1148 ofSEQ ID NO: 27. In other aspects of this embodiment, a binding domaincomprising a BoNT/A NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1139-1148 ofSEQ ID NO: 27. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/A NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino acidadditions relative to amino acids 1098-1110 or amino acids 1139-1148 ofSEQ ID NO: 27. In other aspects of this embodiment, a binding domaincomprising a BoNT/A NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 1098-1110 or amino acids 1139-1148 ofSEQ ID NO: 27.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/B NTNH of SEQ ID NO: 28. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/B NTNHcomprises amino acids 1049-1198 of SEQ ID NO: 28. In another aspect ofthis embodiment, a binding domain comprising a β-trefoil domain derivedfrom a BoNT/B NTNH comprises a α-fold motif of a β-trefoil domain of aBoNT/B NTNH, a β-fold motif of a β-trefoil domain of a BoNT/B NTNH or aγ-fold motif of a β-trefoil domain of a BoNT/B NTNH of SEQ ID NO: 24. Inanother aspect of this embodiment, a β-trefoil domain derived from aBoNT/B NTNH comprises amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1198 of SEQ ID NO: 28.

In other aspects of this embodiment, a binding domain comprising aBoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28, at least 75% aminoacid identity with amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1198 of SEQ ID NO: 28, at least 80% amino acid identitywith amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1198 of SEQ ID NO: 28, at least 85% amino acid identity with aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQID NO: 28, at least 90% amino acid identity with amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28 or atleast 95% amino acid identity with amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. In yet otheraspects of this embodiment, a binding domain comprising a BoNT/B NTNHβ-trefoil domain comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1198 of SEQ ID NO: 28, at most 75% amino acid identitywith amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1198 of SEQ ID NO: 28, at most 80% amino acid identity with aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQID NO: 28, at most 85% amino acid identity with amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28, atmost 90% amino acid identity with amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28 or at most 95%amino acid identity with amino acids 1049-1096, amino acids 1110-1138,or amino acids 1148-1198 of SEQ ID NO: 28.

In other aspects of this embodiment, a binding domain comprising aBoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO:28. In other aspects of this embodiment, a binding domain comprising aBoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO:28. In yet other aspects of this embodiment, a binding domain comprisinga BoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. Inother aspects of this embodiment, a binding domain comprising a BoNT/BNTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. Instill other aspects of this embodiment, a binding domain comprising aBoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. Inother aspects of this embodiment, a binding domain comprising a BoNT/BNTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28.

In other aspects of this embodiment, a binding domain comprising aBoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. Inother aspects of this embodiment, a binding domain comprising a BoNT/BNTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. In yetother aspects of this embodiment, a binding domain comprising a BoNT/BNTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1198 of SEQ ID NO: 28. In other aspectsof this embodiment, a binding domain comprising a BoNT/B NTNH β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1198 of SEQ ID NO: 28. In still other aspects of thisembodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1198 of SEQ ID NO: 28. In other aspects of this embodiment, abinding domain comprising a BoNT/B NTNH β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198of SEQ ID NO: 28.

In another embodiment, a binding domain comprising a BoNT/B NTNHβ-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain ofa BoNT/B NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/BNTNH of SEQ ID NO: 24. In another aspect of this embodiment, a bindingdomain comprising a BoNT/B NTNH β-trefoil domain comprises amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28.

In other aspects of this embodiment, a binding domain comprising aBoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 28, at least 75% amino acid identity with aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, at least 80%amino acid identity with amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 28, at least 85% amino acid identity with amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, at least 90% aminoacid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQID NO: 28 or at least 95% amino acid identity with amino acids 1097-1109or amino acids 1139-1147 of SEQ ID NO: 28. In yet other aspects of thisembodiment, a binding domain comprising a BoNT/B NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, atmost 75% amino acid identity with amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 28, at most 80% amino acid identity with aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, at most 85%amino acid identity with amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 28, at most 90% amino acid identity with amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28 or at most 95% aminoacid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQID NO: 28.

In other aspects of this embodiment, a binding domain comprising aBoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO:28. In other aspects of this embodiment, a binding domain comprising aBoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO:28. In other aspects of this embodiment, a non-contiguous amino acidsubstitution of any amino acid from amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 28 can be replaced with glycine. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany hydrophobic amino acid from amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 28 can be replaced with phenylalanine. In yetother aspects of this embodiment, a binding domain comprising a BoNT/BNTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In otheraspects of this embodiment, a binding domain comprising a BoNT/B NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In stillother aspects of this embodiment, a binding domain comprising a BoNT/BNTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid additions relative to aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In otheraspects of this embodiment, a binding domain comprising a BoNT/B NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28.

In other aspects of this embodiment, a binding domain comprising aBoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO:28. In other aspects of this embodiment, a binding domain comprising aBoNT/B NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO:28. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 28 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 28 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a BoNT/B NTNH comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino aciddeletions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 28. In other aspects of this embodiment, a binding domaincomprising a BoNT/B NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 28. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/B NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino acidadditions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 28. In other aspects of this embodiment, a binding domaincomprising a BoNT/B NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 28.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/C1 NTNH of SEQ ID NO: 29. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/C1 NTNHcomprises amino acids 1049-1197 of SEQ ID NO: 29. In another aspect ofthis embodiment, a binding domain comprising a β-trefoil domain derivedfrom a BoNT/C1 NTNH comprises a α-fold motif of a β-trefoil domain of aBoNT/C1 NTNH, a β-fold motif of a β-trefoil domain of a BoNT/C1 NTNH ora γ-fold motif of a β-trefoil domain of a BoNT/C1 NTNH of SEQ ID NO: 24.In another aspect of this embodiment, a β-trefoil domain derived from aBoNT/C1 NTNH comprises amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1197 of SEQ ID NO: 29.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29, at least 75% aminoacid identity with amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1197 of SEQ ID NO: 29, at least 80% amino acid identitywith amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1197 of SEQ ID NO: 29, at least 85% amino acid identity with aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQID NO: 29, at least 90% amino acid identity with amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29 or atleast 95% amino acid identity with amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. In yet otheraspects of this embodiment, a binding domain comprising a BoNT/C1 NTNHβ-trefoil domain comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1197 of SEQ ID NO: 29, at most 75% amino acid identitywith amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1197 of SEQ ID NO: 29, at most 80% amino acid identity with aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQID NO: 29, at most 85% amino acid identity with amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29, atmost 90% amino acid identity with amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29 or at most 95%amino acid identity with amino acids 1049-1096, amino acids 1110-1138,or amino acids 1148-1197 of SEQ ID NO: 29.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO:29. In other aspects of this embodiment, a binding domain comprising aBoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO:29. In yet other aspects of this embodiment, a binding domain comprisinga BoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. Inother aspects of this embodiment, a binding domain comprising a BoNT/C1NTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. Instill other aspects of this embodiment, a binding domain comprising aBoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. Inother aspects of this embodiment, a binding domain comprising a BoNT/C1NTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. Inother aspects of this embodiment, a binding domain comprising a BoNT/C1NTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. In yetother aspects of this embodiment, a binding domain comprising a BoNT/C1NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1197 of SEQ ID NO: 29. In other aspectsof this embodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1197 of SEQ ID NO: 29. In still other aspects of thisembodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1197 of SEQ ID NO: 29. In other aspects of this embodiment, abinding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197of SEQ ID NO: 29.

In another embodiment, a binding domain comprising a BoNT/C1 NTNHβ-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain ofa BoNT/C1 NTNH or a β8/β9 hairpin turn of a β-trefoil domain of aBoNT/C1 NTNH of SEQ ID NO: 24. In another aspect of this embodiment, abinding domain comprising a BoNT/C1 NTNH β-trefoil domain comprisesamino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 29, at least 75% amino acid identity with aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29, at least 80%amino acid identity with amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 29, at least 85% amino acid identity with amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29, at least 90% aminoacid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQID NO: 29 or at least 95% amino acid identity with amino acids 1097-1109or amino acids 1139-1147 of SEQ ID NO: 29. In yet other aspects of thisembodiment, a binding domain comprising a BoNT/C1 NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29, atmost 75% amino acid identity with amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 29, at most 80% amino acid identity with aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29, at most 85%amino acid identity with amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 29, at most 90% amino acid identity with amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29 or at most 95% aminoacid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQID NO: 29.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO:29. In other aspects of this embodiment, a binding domain comprising aBoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO:29. In other aspects of this embodiment, a non-contiguous amino acidsubstitution of any amino acid from amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 29 can be replaced with glycine. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany hydrophobic amino acid from amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 29 can be replaced with phenylalanine. In yetother aspects of this embodiment, a binding domain comprising a BoNT/C1NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29. In otheraspects of this embodiment, a binding domain comprising a BoNT/C1 NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29. In stillother aspects of this embodiment, a binding domain comprising a BoNT/C1NTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid additions relative to aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29. In otheraspects of this embodiment, a binding domain comprising a BoNT/C1 NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 29.

In other aspects of this embodiment, a binding domain comprising aBoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO:29. In other aspects of this embodiment, a binding domain comprising aBoNT/C1 NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO:29. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 29 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 29 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a BoNT/C1 NTNH comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino aciddeletions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 29. In other aspects of this embodiment, a binding domaincomprising a BoNT/C1 NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 29. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/C1 NTNH β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four contiguousamino acid additions relative to amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 29. In other aspects of this embodiment, abinding domain comprising a BoNT/C1 NTNH β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid additions relative to amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 29.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/D NTNH of SEQ ID NO: 30. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/D NTNHcomprises amino acids 1049-1197 of SEQ ID NO: 30. In another aspect ofthis embodiment, a binding domain comprising a β-trefoil domain derivedfrom a BoNT/D NTNH comprises a α-fold motif of a β-trefoil domain of aBoNT/D NTNH, a β-fold motif of a β-trefoil domain of a BoNT/D NTNH or aγ-fold motif of a β-trefoil domain of a BoNT/D NTNH of SEQ ID NO: 24. Inanother aspect of this embodiment, a β-trefoil domain derived from aBoNT/D NTNH comprises amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1197 of SEQ ID NO: 30.

In other aspects of this embodiment, a binding domain comprising aBoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30, at least 75% aminoacid identity with amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1197 of SEQ ID NO: 30, at least 80% amino acid identitywith amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1197 of SEQ ID NO: 30, at least 85% amino acid identity with aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQID NO: 30, at least 90% amino acid identity with amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30 or atleast 95% amino acid identity with amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. In yet otheraspects of this embodiment, a binding domain comprising a BoNT/D NTNHβ-trefoil domain comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1197 of SEQ ID NO: 30, at most 75% amino acid identitywith amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1197 of SEQ ID NO: 30, at most 80% amino acid identity with aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQID NO: 30, at most 85% amino acid identity with amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30, atmost 90% amino acid identity with amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30 or at most 95%amino acid identity with amino acids 1049-1096, amino acids 1110-1138,or amino acids 1148-1197 of SEQ ID NO: 30.

In other aspects of this embodiment, a binding domain comprising aBoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO:30. In other aspects of this embodiment, a binding domain comprising aBoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO:30. In yet other aspects of this embodiment, a binding domain comprisinga BoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. Inother aspects of this embodiment, a binding domain comprising a BoNT/DNTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. Instill other aspects of this embodiment, a binding domain comprising aBoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. Inother aspects of this embodiment, a binding domain comprising a BoNT/DNTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30.

In other aspects of this embodiment, a binding domain comprising aBoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. Inother aspects of this embodiment, a binding domain comprising a BoNT/DNTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. In yetother aspects of this embodiment, a binding domain comprising a BoNT/DNTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1197 of SEQ ID NO: 30. In other aspectsof this embodiment, a binding domain comprising a BoNT/D NTNH β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1197 of SEQ ID NO: 30. In still other aspects of thisembodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1197 of SEQ ID NO: 30. In other aspects of this embodiment, abinding domain comprising a BoNT/D NTNH β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197of SEQ ID NO: 30.

In another embodiment, a binding domain comprising a BoNT/D NTNHβ-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain ofa BoNT/D NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/DNTNH of SEQ ID NO: 24. In another aspect of this embodiment, a bindingdomain comprising a BoNT/D NTNH β-trefoil domain comprises amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30.

In other aspects of this embodiment, a binding domain comprising aBoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 30, at least 75% amino acid identity with aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30, at least 80%amino acid identity with amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 30, at least 85% amino acid identity with amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30, at least 90% aminoacid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQID NO: 30 or at least 95% amino acid identity with amino acids 1097-1109or amino acids 1139-1147 of SEQ ID NO: 30. In yet other aspects of thisembodiment, a binding domain comprising a BoNT/D NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30, atmost 75% amino acid identity with amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 30, at most 80% amino acid identity with aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30, at most 85%amino acid identity with amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 30, at most 90% amino acid identity with amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30 or at most 95% aminoacid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQID NO: 30.

In other aspects of this embodiment, a binding domain comprising aBoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO:30. In other aspects of this embodiment, a binding domain comprising aBoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO:30. In other aspects of this embodiment, a non-contiguous amino acidsubstitution of any amino acid from amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 30 can be replaced with glycine. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany hydrophobic amino acid from amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 30 can be replaced with phenylalanine. In yetother aspects of this embodiment, a binding domain comprising a BoNT/DNTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30. In otheraspects of this embodiment, a binding domain comprising a BoNT/D NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30. In stillother aspects of this embodiment, a binding domain comprising a BoNT/DNTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid additions relative to aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30. In otheraspects of this embodiment, a binding domain comprising a BoNT/D NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 30.

In other aspects of this embodiment, a binding domain comprising aBoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO:30. In other aspects of this embodiment, a binding domain comprising aBoNT/D NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO:30. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 30 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 30 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a BoNT/D NTNH comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino aciddeletions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 30. In other aspects of this embodiment, a binding domaincomprising a BoNT/D NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 30. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/D NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino acidadditions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 30. In other aspects of this embodiment, a binding domaincomprising a BoNT/D NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 30.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/E NTNH of SEQ ID NO: 31. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/E NTNHcomprises amino acids 1014-1163 of SEQ ID NO: 31. In another aspect ofthis embodiment, a binding domain comprising a β-trefoil domain derivedfrom a BoNT/E NTNH comprises a α-fold motif of a β-trefoil domain of aBoNT/E NTNH, a β-fold motif of a β-trefoil domain of a BoNT/E NTNH or aγ-fold motif of a β-trefoil domain of a BoNT/E NTNH of SEQ ID NO: 24. Inanother aspect of this embodiment, a β-trefoil domain derived from aBoNT/E NTNH comprises amino acids 1014-1061, amino acids 1075-1103, oramino acids 1114-1163 of SEQ ID NO: 31.

In other aspects of this embodiment, a binding domain comprising aBoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1014-1061, amino acids1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31, at least 75% aminoacid identity with amino acids 1014-1061, amino acids 1075-1103, oramino acids 1114-1163 of SEQ ID NO: 31, at least 80% amino acid identitywith amino acids 1014-1061, amino acids 1075-1103, or amino acids1114-1163 of SEQ ID NO: 31, at least 85% amino acid identity with aminoacids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQID NO: 31, at least 90% amino acid identity with amino acids 1014-1061,amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31 or atleast 95% amino acid identity with amino acids 1014-1061, amino acids1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. In yet otheraspects of this embodiment, a binding domain comprising a BoNT/E NTNHβ-trefoil domain comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1014-1061, amino acids 1075-1103, oramino acids 1114-1163 of SEQ ID NO: 31, at most 75% amino acid identitywith amino acids 1014-1061, amino acids 1075-1103, or amino acids1114-1163 of SEQ ID NO: 31, at most 80% amino acid identity with aminoacids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQID NO: 31, at most 85% amino acid identity with amino acids 1014-1061,amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31, atmost 90% amino acid identity with amino acids 1014-1061, amino acids1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31 or at most 95%amino acid identity with amino acids 1014-1061, amino acids 1075-1103,or amino acids 1114-1163 of SEQ ID NO: 31.

In other aspects of this embodiment, a binding domain comprising aBoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO:31. In other aspects of this embodiment, a binding domain comprising aBoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO:31. In yet other aspects of this embodiment, a binding domain comprisinga BoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1014-1061,amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. Inother aspects of this embodiment, a binding domain comprising a BoNT/ENTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1014-1061,amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. Instill other aspects of this embodiment, a binding domain comprising aBoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1014-1061,amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. Inother aspects of this embodiment, a binding domain comprising a BoNT/ENTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1014-1061,amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31.

In other aspects of this embodiment, a binding domain comprising aBoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1014-1061,amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. Inother aspects of this embodiment, a binding domain comprising a BoNT/ENTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1014-1061,amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. In yetother aspects of this embodiment, a binding domain comprising a BoNT/ENTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 1014-1061, amino acids1075-1103, or amino acids 1114-1163 of SEQ ID NO: 31. In other aspectsof this embodiment, a binding domain comprising a BoNT/E NTNH β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 1014-1061, amino acids 1075-1103, oramino acids 1114-1163 of SEQ ID NO: 31. In still other aspects of thisembodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1014-1061, amino acids 1075-1103, or amino acids1114-1163 of SEQ ID NO: 31. In other aspects of this embodiment, abinding domain comprising a BoNT/E NTNH β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163of SEQ ID NO: 31.

In another embodiment, a binding domain comprising a BoNT/E NTNHβ-trefoil domain comprises a β4/β5 hairpin turn of β-trefoil domain of aBoNT/E NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/ENTNH of SEQ ID NO: 24. In another aspect of this embodiment, a bindingdomain comprising a BoNT/E NTNH β-trefoil domain comprises amino acids1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31.

In other aspects of this embodiment, a binding domain comprising aBoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1062-1074 or amino acids1104-1113 of SEQ ID NO: 31, at least 75% amino acid identity with aminoacids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31, at least 80%amino acid identity with amino acids 1062-1074 or amino acids 1104-1113of SEQ ID NO: 31, at least 85% amino acid identity with amino acids1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31, at least 90% aminoacid identity with amino acids 1062-1074 or amino acids 1104-1113 of SEQID NO: 31 or at least 95% amino acid identity with amino acids 1062-1074or amino acids 1104-1113 of SEQ ID NO: 31. In yet other aspects of thisembodiment, a binding domain comprising a BoNT/E NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31, atmost 75% amino acid identity with amino acids 1062-1074 or amino acids1104-1113 of SEQ ID NO: 31, at most 80% amino acid identity with aminoacids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31, at most 85%amino acid identity with amino acids 1062-1074 or amino acids 1104-1113of SEQ ID NO: 31, at most 90% amino acid identity with amino acids1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31 or at most 95% aminoacid identity with amino acids 1062-1074 or amino acids 1104-1113 of SEQID NO: 31.

In other aspects of this embodiment, a binding domain comprising aBoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO:31. In other aspects of this embodiment, a binding domain comprising aBoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO:31. In other aspects of this embodiment, a non-contiguous amino acidsubstitution of any amino acid from amino acids 1062-1074 or amino acids1104-1113 of SEQ ID NO: 31 can be replaced with glycine. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany hydrophobic amino acid from amino acids 1062-1074 or amino acids1104-1113 of SEQ ID NO: 31 can be replaced with phenylalanine. In yetother aspects of this embodiment, a binding domain comprising a BoNT/ENTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31. In otheraspects of this embodiment, a binding domain comprising a BoNT/E NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31. In stillother aspects of this embodiment, a binding domain comprising a BoNT/ENTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid additions relative to aminoacids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31. In otheraspects of this embodiment, a binding domain comprising a BoNT/E NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 31.

In other aspects of this embodiment, a binding domain comprising aBoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO:31. In other aspects of this embodiment, a binding domain comprising aBoNT/E NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO:31. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 1062-1074 or amino acids1104-1113 of SEQ ID NO: 31 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1062-1074 or amino acids1104-1113 of SEQ ID NO: 31 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a BoNT/E NTNH comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino aciddeletions relative to amino acids 1062-1074 or amino acids 1104-1113 ofSEQ ID NO: 31. In other aspects of this embodiment, a binding domaincomprising a BoNT/E NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1062-1074 or amino acids 1104-1113 ofSEQ ID NO: 31. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/E NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino acidadditions relative to amino acids 1062-1074 or amino acids 1104-1113 ofSEQ ID NO: 31. In other aspects of this embodiment, a binding domaincomprising a BoNT/E NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 1062-1074 or amino acids 1104-1113 ofSEQ ID NO: 31.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/F NTNH of SEQ ID NO: 32. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/F NTNHcomprises amino acids 1016-1160 of SEQ ID NO: 32. In another aspect ofthis embodiment, a binding domain comprising a β-trefoil domain derivedfrom a BoNT/F NTNH comprises a α-fold motif of a β-trefoil domain of aBoNT/F NTNH, a β-fold motif of a β-trefoil domain of a BoNT/F NTNH or aγ-fold motif of a β-trefoil domain of a BoNT/F NTNH of SEQ ID NO: 24. Inanother aspect of this embodiment, a β-trefoil domain derived from aBoNT/F NTNH comprises amino acids 1016-1063, amino acids 1077-1104, oramino acids 1115-1160 of SEQ ID NO: 32.

In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1016-1063, amino acids1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32, at least 75% aminoacid identity with amino acids 1016-1063, amino acids 1077-1104, oramino acids 1115-1160 of SEQ ID NO: 32, at least 80% amino acid identitywith amino acids 1016-1063, amino acids 1077-1104, or amino acids1115-1160 of SEQ ID NO: 32, at least 85% amino acid identity with aminoacids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQID NO: 32, at least 90% amino acid identity with amino acids 1016-1063,amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32 or atleast 95% amino acid identity with amino acids 1016-1063, amino acids1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. In yet otheraspects of this embodiment, a binding domain comprising a BoNT/F NTNHβ-trefoil domain comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1016-1063, amino acids 1077-1104, oramino acids 1115-1160 of SEQ ID NO: 32, at most 75% amino acid identitywith amino acids 1016-1063, amino acids 1077-1104, or amino acids1115-1160 of SEQ ID NO: 32, at most 80% amino acid identity with aminoacids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQID NO: 32, at most 85% amino acid identity with amino acids 1016-1063,amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32, atmost 90% amino acid identity with amino acids 1016-1063, amino acids1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32 or at most 95%amino acid identity with amino acids 1016-1063, amino acids 1077-1104,or amino acids 1115-1160 of SEQ ID NO: 32.

In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO:32. In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO:32. In yet other aspects of this embodiment, a binding domain comprisinga BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1016-1063,amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. Inother aspects of this embodiment, a binding domain comprising a BoNT/FNTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1016-1063,amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. Instill other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1016-1063,amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. Inother aspects of this embodiment, a binding domain comprising a BoNT/FNTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1016-1063,amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32.

In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1016-1063,amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. Inother aspects of this embodiment, a binding domain comprising a BoNT/FNTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1016-1063,amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. In yetother aspects of this embodiment, a binding domain comprising a BoNT/FNTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 1016-1063, amino acids1077-1104, or amino acids 1115-1160 of SEQ ID NO: 32. In other aspectsof this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 1016-1063, amino acids 1077-1104, oramino acids 1115-1160 of SEQ ID NO: 32. In still other aspects of thisembodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1016-1063, amino acids 1077-1104, or amino acids1115-1160 of SEQ ID NO: 32. In other aspects of this embodiment, abinding domain comprising a BoNT/F NTNH β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160of SEQ ID NO: 32.

In another embodiment, a binding domain comprising a BoNT/F NTNHβ-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain ofa BoNT/F NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/FNTNH of SEQ ID NO: 24. In another aspect of this embodiment, a bindingdomain comprising a BoNT/F NTNH β-trefoil domain comprises amino acids1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32.

In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1064-1076 or amino acids1105-1114 of SEQ ID NO: 32, at least 75% amino acid identity with aminoacids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32, at least 80%amino acid identity with amino acids 1064-1076 or amino acids 1105-1114of SEQ ID NO: 32, at least 85% amino acid identity with amino acids1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32, at least 90% aminoacid identity with amino acids 1064-1076 or amino acids 1105-1114 of SEQID NO: 32 or at least 95% amino acid identity with amino acids 1064-1076or amino acids 1105-1114 of SEQ ID NO: 32. In yet other aspects of thisembodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32, atmost 75% amino acid identity with amino acids 1064-1076 or amino acids1105-1114 of SEQ ID NO: 32, at most 80% amino acid identity with aminoacids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32, at most 85%amino acid identity with amino acids 1064-1076 or amino acids 1105-1114of SEQ ID NO: 32, at most 90% amino acid identity with amino acids1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32 or at most 95% aminoacid identity with amino acids 1064-1076 or amino acids 1105-1114 of SEQID NO: 32.

In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO:32. In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO:32. In other aspects of this embodiment, a non-contiguous amino acidsubstitution of any amino acid from amino acids 1064-1076 or amino acids1105-1114 of SEQ ID NO: 32 can be replaced with glycine. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany hydrophobic amino acid from amino acids 1064-1076 or amino acids1105-1114 of SEQ ID NO: 32 can be replaced with phenylalanine. In yetother aspects of this embodiment, a binding domain comprising a BoNT/FNTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32. In otheraspects of this embodiment, a binding domain comprising a BoNT/F NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32. In stillother aspects of this embodiment, a binding domain comprising a BoNT/FNTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid additions relative to aminoacids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32. In otheraspects of this embodiment, a binding domain comprising a BoNT/F NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 32.

In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO:32. In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO:32. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 1064-1076 or amino acids1105-1114 of SEQ ID NO: 32 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1064-1076 or amino acids1105-1114 of SEQ ID NO: 32 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a BoNT/F NTNH comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino aciddeletions relative to amino acids 1064-1076 or amino acids 1105-1114 ofSEQ ID NO: 32. In other aspects of this embodiment, a binding domaincomprising a BoNT/F NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1064-1076 or amino acids 1105-1114 ofSEQ ID NO: 32. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino acidadditions relative to amino acids 1064-1076 or amino acids 1105-1114 ofSEQ ID NO: 32. In other aspects of this embodiment, a binding domaincomprising a BoNT/F NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 1064-1076 or amino acids 1105-1114 ofSEQ ID NO: 32.

In another embodiment, a binding domain comprises a β-trefoil domainderived from a BoNT/F NTNH of SEQ ID NO: 33. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/F NTNHcomprises amino acids 1017-1166 of SEQ ID NO: 33. In another aspect ofthis embodiment, a binding domain comprising a β-trefoil domain derivedfrom a BoNT/F NTNH comprises a α-fold motif of a β-trefoil domain of aBoNT/F NTNH, a β-fold motif of a β-trefoil domain of a BoNT/F NTNH or aγ-fold motif of a β-trefoil domain of a BoNT/F NTNH of SEQ ID NO: 24. Inanother aspect of this embodiment, a β-trefoil domain derived from aBoNT/F NTNH comprises amino acids 1017-1064, amino acids 1078-1106, oramino acids 1117-1166 of SEQ ID NO: 33.

In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1017-1064, amino acids1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33, at least 75% aminoacid identity with amino acids 1017-1064, amino acids 1078-1106, oramino acids 1117-1166 of SEQ ID NO: 33, at least 80% amino acid identitywith amino acids 1017-1064, amino acids 1078-1106, or amino acids1117-1166 of SEQ ID NO: 33, at least 85% amino acid identity with aminoacids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQID NO: 33, at least 90% amino acid identity with amino acids 1017-1064,amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33 or atleast 95% amino acid identity with amino acids 1017-1064, amino acids1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. In yet otheraspects of this embodiment, a binding domain comprising a BoNT/F NTNHβ-trefoil domain comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1017-1064, amino acids 1078-1106, oramino acids 1117-1166 of SEQ ID NO: 33, at most 75% amino acid identitywith amino acids 1017-1064, amino acids 1078-1106, or amino acids1117-1166 of SEQ ID NO: 33, at most 80% amino acid identity with aminoacids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQID NO: 33, at most 85% amino acid identity with amino acids 1017-1064,amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33, atmost 90% amino acid identity with amino acids 1017-1064, amino acids1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33 or at most 95%amino acid identity with amino acids 1017-1064, amino acids 1078-1106,or amino acids 1117-1166 of SEQ ID NO: 33.

In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO:33. In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO:33. In yet other aspects of this embodiment, a binding domain comprisinga BoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1017-1064,amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. Inother aspects of this embodiment, a binding domain comprising a BoNT/FNTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1017-1064,amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. Instill other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1017-1064,amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. Inother aspects of this embodiment, a binding domain comprising a BoNT/FNTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1017-1064,amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33.

In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1017-1064,amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. Inother aspects of this embodiment, a binding domain comprising a BoNT/FNTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1017-1064,amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. In yetother aspects of this embodiment, a binding domain comprising a BoNT/FNTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 1017-1064, amino acids1078-1106, or amino acids 1117-1166 of SEQ ID NO: 33. In other aspectsof this embodiment, a binding domain comprising a BoNT/F NTNH β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 1017-1064, amino acids 1078-1106, oramino acids 1117-1166 of SEQ ID NO: 33. In still other aspects of thisembodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1017-1064, amino acids 1078-1106, or amino acids1117-1166 of SEQ ID NO: 33. In other aspects of this embodiment, abinding domain comprising a BoNT/F NTNH β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166of SEQ ID NO: 33.

In another embodiment, a binding domain comprising a BoNT/F NTNHβ-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain ofa BoNT/F NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/FNTNH of SEQ ID NO: 24. In another aspect of this embodiment, a bindingdomain comprising a BoNT/F NTNH β-trefoil domain comprises amino acids1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33.

In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1065-1077 or amino acids1107-1116 of SEQ ID NO: 33, at least 75% amino acid identity with aminoacids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33, at least 80%amino acid identity with amino acids 1065-1077 or amino acids 1107-1116of SEQ ID NO: 33, at least 85% amino acid identity with amino acids1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33, at least 90% aminoacid identity with amino acids 1065-1077 or amino acids 1107-1116 of SEQID NO: 33 or at least 95% amino acid identity with amino acids 1065-1077or amino acids 1107-1116 of SEQ ID NO: 33. In yet other aspects of thisembodiment, a binding domain comprising a BoNT/F NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33, atmost 75% amino acid identity with amino acids 1065-1077 or amino acids1107-1116 of SEQ ID NO: 33, at most 80% amino acid identity with aminoacids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33, at most 85%amino acid identity with amino acids 1065-1077 or amino acids 1107-1116of SEQ ID NO: 33, at most 90% amino acid identity with amino acids1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33 or at most 95% aminoacid identity with amino acids 1065-1077 or amino acids 1107-1116 of SEQID NO: 33.

In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO:33. In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO:33. In other aspects of this embodiment, a non-contiguous amino acidsubstitution of any amino acid from amino acids 1065-1077 or amino acids1107-1116 of SEQ ID NO: 33 can be replaced with glycine. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany hydrophobic amino acid from amino acids 1065-1077 or amino acids1107-1116 of SEQ ID NO: 33 can be replaced with phenylalanine. In yetother aspects of this embodiment, a binding domain comprising a BoNT/FNTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33. In otheraspects of this embodiment, a binding domain comprising a BoNT/F NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33. In stillother aspects of this embodiment, a binding domain comprising a BoNT/FNTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid additions relative to aminoacids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33. In otheraspects of this embodiment, a binding domain comprising a BoNT/F NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 33.

In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO:33. In other aspects of this embodiment, a binding domain comprising aBoNT/F NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO:33. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 1065-1077 or amino acids1107-1116 of SEQ ID NO: 33 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1065-1077 or amino acids1107-1116 of SEQ ID NO: 33 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a BoNT/F NTNH comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino aciddeletions relative to amino acids 1065-1077 or amino acids 1107-1116 ofSEQ ID NO: 33. In other aspects of this embodiment, a binding domaincomprising a BoNT/F NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1065-1077 or amino acids 1107-1116 ofSEQ ID NO: 33. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/F NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino acidadditions relative to amino acids 1065-1077 or amino acids 1107-1116 ofSEQ ID NO: 33. In other aspects of this embodiment, a binding domaincomprising a BoNT/F NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 1065-1077 or amino acids 1107-1116 ofSEQ ID NO: 33.

In another embodiment, a binding domain comprising a β-trefoil domainderived from a BoNT/G NTNH of SEQ ID NO: 34. In another embodiment, abinding domain comprising a β-trefoil domain derived from a BoNT/G NTNHcomprises amino acids 1050-1199 of SEQ ID NO: 34. In another aspect ofthis embodiment, a binding domain comprising a β-trefoil domain derivedfrom a BoNT/G NTNH comprises a α-fold motif of a β-trefoil domain of aBoNT/G NTNH, a β-fold motif of a β-trefoil domain of a BoNT/G NTNH or aγ-fold motif of a β-trefoil domain of a BoNT/G NTNH of SEQ ID NO: 24. Inanother aspect of this embodiment, a β-trefoil domain derived from aBoNT/G NTNH comprises amino acids 1050-1097, amino acids 1111-1139, oramino acids 1150-1199 of SEQ ID NO: 34.

In other aspects of this embodiment, a binding domain comprising aBoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1050-1097, amino acids1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34, at least 75% aminoacid identity with amino acids 1050-1097, amino acids 1111-1139, oramino acids 1150-1199 of SEQ ID NO: 34, at least 80% amino acid identitywith amino acids 1050-1097, amino acids 1111-1139, or amino acids1150-1199 of SEQ ID NO: 34, at least 85% amino acid identity with aminoacids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQID NO: 34, at least 90% amino acid identity with amino acids 1050-1097,amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34 or atleast 95% amino acid identity with amino acids 1050-1097, amino acids1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. In yet otheraspects of this embodiment, a binding domain comprising a BoNT/G NTNHβ-trefoil domain comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1050-1097, amino acids 1111-1139, oramino acids 1150-1199 of SEQ ID NO: 34, at most 75% amino acid identitywith amino acids 1050-1097, amino acids 1111-1139, or amino acids1150-1199 of SEQ ID NO: 34, at most 80% amino acid identity with aminoacids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQID NO: 34, at most 85% amino acid identity with amino acids 1050-1097,amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34, atmost 90% amino acid identity with amino acids 1050-1097, amino acids1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34 or at most 95%amino acid identity with amino acids 1050-1097, amino acids 1111-1139,or amino acids 1150-1199 of SEQ ID NO: 34.

In other aspects of this embodiment, a binding domain comprising aBoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO:34. In other aspects of this embodiment, a binding domain comprising aBoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO:34. In yet other aspects of this embodiment, a binding domain comprisinga BoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1050-1097,amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. Inother aspects of this embodiment, a binding domain comprising a BoNT/GNTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 1050-1097,amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. Instill other aspects of this embodiment, a binding domain comprising aBoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1050-1097,amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. Inother aspects of this embodiment, a binding domain comprising a BoNT/GNTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1050-1097,amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34.

In other aspects of this embodiment, a binding domain comprising aBoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1050-1097,amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. Inother aspects of this embodiment, a binding domain comprising a BoNT/GNTNH β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 1050-1097,amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. In yetother aspects of this embodiment, a binding domain comprising a BoNT/GNTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 1050-1097, amino acids1111-1139, or amino acids 1150-1199 of SEQ ID NO: 34. In other aspectsof this embodiment, a binding domain comprising a BoNT/G NTNH β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 1050-1097, amino acids 1111-1139, oramino acids 1150-1199 of SEQ ID NO: 34. In still other aspects of thisembodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1050-1097, amino acids 1111-1139, or amino acids1150-1199 of SEQ ID NO: 34. In other aspects of this embodiment, abinding domain comprising a BoNT/G NTNH β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199of SEQ ID NO: 34.

In another embodiment, a binding domain comprising a BoNT/G NTNHβ-trefoil domain comprises a β4/β5 hairpin turn of a β-trefoil domain ofa BoNT/G NTNH or a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/GNTNH of SEQ ID NO: 24. In another aspect of this embodiment, a bindingdomain comprising a BoNT/G NTNH β-trefoil domain comprises amino acids1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34.

In other aspects of this embodiment, a binding domain comprising aBoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with amino acids 1098-1110 or amino acids1140-1149 of SEQ ID NO: 34, at least 75% amino acid identity with aminoacids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34, at least 80%amino acid identity with amino acids 1098-1110 or amino acids 1140-1149of SEQ ID NO: 34, at least 85% amino acid identity with amino acids1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34, at least 90% aminoacid identity with amino acids 1098-1110 or amino acids 1140-1149 of SEQID NO: 34 or at least 95% amino acid identity with amino acids 1098-1110or amino acids 1140-1149 of SEQ ID NO: 34. In yet other aspects of thisembodiment, a binding domain comprising a BoNT/G NTNH β-trefoil domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34, atmost 75% amino acid identity with amino acids 1098-1110 or amino acids1140-1149 of SEQ ID NO: 34, at most 80% amino acid identity with aminoacids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34, at most 85%amino acid identity with amino acids 1098-1110 or amino acids 1140-1149of SEQ ID NO: 34, at most 90% amino acid identity with amino acids1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34 or at most 95% aminoacid identity with amino acids 1098-1110 or amino acids 1140-1149 of SEQID NO: 34.

In other aspects of this embodiment, a binding domain comprising aBoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO:34. In other aspects of this embodiment, a binding domain comprising aBoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four non-contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO:34. In other aspects of this embodiment, a non-contiguous amino acidsubstitution of any amino acid from amino acids 1098-1110 or amino acids1140-1149 of SEQ ID NO: 34 can be replaced with glycine. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany hydrophobic amino acid from amino acids 1098-1110 or amino acids1140-1149 of SEQ ID NO: 34 can be replaced with phenylalanine. In yetother aspects of this embodiment, a binding domain comprising a BoNT/GNTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34. In otheraspects of this embodiment, a binding domain comprising a BoNT/G NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34. In stillother aspects of this embodiment, a binding domain comprising a BoNT/GNTNH β-trefoil domain comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid additions relative to aminoacids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34. In otheraspects of this embodiment, a binding domain comprising a BoNT/G NTNHβ-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid additions relative to aminoacids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 34.

In other aspects of this embodiment, a binding domain comprising aBoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., atmost one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO:34. In other aspects of this embodiment, a binding domain comprising aBoNT/G NTNH β-trefoil domain comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid substitutionsrelative to amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO:34. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 1098-1110 or amino acids1140-1149 of SEQ ID NO: 34 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1098-1110 or amino acids1140-1149 of SEQ ID NO: 34 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a BoNT/G NTNH comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1140-1149 ofSEQ ID NO: 34. In other aspects of this embodiment, a binding domaincomprising a BoNT/G NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1140-1149 ofSEQ ID NO: 34. In still other aspects of this embodiment, a bindingdomain comprising a BoNT/G NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino acidadditions relative to amino acids 1098-1110 or amino acids 1140-1149 ofSEQ ID NO: 34. In other aspects of this embodiment, a binding domaincomprising a BoNT/G NTNH β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 1098-1110 or amino acids 1140-1149 ofSEQ ID NO: 34.

Another example of a binding domain, includes, without limitation,ligands that bind the same receptors as does the naturally-occurringClostridial toxins. Recent studies are revealing components of theendogenous receptors used by a Clostridial toxin during the intoxicationprocess. As a non-limiting example, fibroblast growth factor 3 receptor(FGFR3) serves as a BoNT/A receptor, see, e.g., Ester Fernandez-Salas etal., Botulinum Toxin Screening Assays, PCT Patent Application No.2005/006421 (Sep. 9, 2005). As another non-limiting example,synaptotagmin I serves as a BoNT/B receptor and as a BoNT/G receptor,see, e.g., Min Dong et al., Synaptotagmins I and II mediate entry ofbotulinum neurotoxin B into cells, 162(7) J. Cell Biol. 1293-1303(2003); and Andreas Rummel et al., Synaptotagmins I and II act as nervecell receptors for botulinum neurotoxin G, 279(29) J. Biol. Chem.30865-30870 (2004). As yet another non-limiting example, synaptotagminII serves as a BoNT/B receptor and as a BoNT/G receptor, see, e.g., MinDong et al., supra, (2003); and Andreas Rummel et al., supra, (2004).The selection of a binding domain will depend on the Clostridial toxinbeing modified. As non-limiting examples, ligands that selectively bindto a FGFR3 could be used as a binding domain for a modified BoNT/A,whereas ligands that selectively bind a Synaptotagmin I or SynaptotagminII could be used as a binding domain for a modified BoNT/B or a modifiedBoNT/G.

In addition to its enhanced targeting activity, replacement of anaturally-occurring binding domain with, e.g., an FGF ligand, has anadded advantage of reducing the likelihood of the modified toxin fromeliciting an immunogenic response. Regions found in the H_(CC) targetingdomain are bound by neutralizing anti-BoNT/A antibodies, see, e.g., M.Zouhair Atassi et al., Mapping of the Antibody-binding Regions onBotulinum Neurotoxin H-chain Domain 855-1296 with Anti-toxin Antibodiesfrom Three Host Species, 15 J. PROT. CHEM. 691-700, (1996); M. ZouhairAtassi & Behzod Z. Dolimbek, Mapping of the Antibody-binding Profile onBotulinum Neurotoxin A H _(N)-domain (residues 449-859) with Anti-toxinAntibodies from Four Host Species. Antigenic Profile of the EntireH-chain of Botulinum Neurotoxin A, 23(1) PROTEIN J. 39-52, (2004).Therefore, elimination of this binding domain will reduce the likelihoodof an immunogenic response because 1) the Clostridial toxin H_(CC)binding domain is absent; 2) a human FGF binding domain will most likelynot elicit an immunogenic response in a patient because it is a humanpolypeptide.

Fibroblast growth factors (FGF) participate in many developmental,differentiation and growth and repair processes of cells through complexcombinatorial signaling pathways. Presently, at least 23 ligands(FGF1-23) are known to signal through a family of five transmembranetyrosine kinase FGF receptors (FGFR1-5). Affinity of FGFRs for theirligands is highly diverse with different affinities for each familymember of growth factors, see, e.g., C. J. Powers et al., Fibroblastgrowth factors, their receptors and signaling, 7(3) Endocr. Relat.Cancer. 165-197 (2000). This diversity is achieved in part by thegeneration of alternatively spliced variants encoding distinct receptorisoforms, see, e.g., Bernhard Reuss & Oliver von Bohlen und Halbach,Fibroblast growth factors and their receptors in the central nervoussystem, 313(2) Cell Tissue Res. 139-157 (2003). The protein region thatappears to have the highest influence on ligand binding selectivity is aportion of the IgIII domain, for which isoforms encoded by threedifferent splice variants have been identified. These three isoforms,designated IgIIIa, IgIIIb and IgIIIc, have relative binding affinitiesfor different FGFR family members. Alternative splicing in the FGFRligand binding domain, designated a and b, generates additional receptorisoforms with novel ligand affinities. Isoforms for IgIIIa, IgIIIb andIgIIIc have been identified for both FGFR1 and FGFR2. Thus far, theIgIIIa isoform of FGFR3 and the IgIIIa and IgIIIb isoforms of FGFR4 andFGFR5 have not been reported.

Currently, several FGFs have been shown to selectively bind FGFR3, suchas, e.g., FGF-1, FGF-2, FGF-4, FGF-8, FGF-9, FGF-17 and FGF-18, see,e.g., Hecht et al., 1995; Ornitz et al., 1996; and Xu et al., 2000.Additional studies have revealed that FGF-1 and FGF9 preferentially bindFGFR3IIIb, whereas FGF-1 FGF-2, FGF-4, FGF-8, and FGF9 preferentiallybind FGFR3IIIc. Studies have shown that each of these ligands is presentin various vertebrate species with a high degree of sequence identitywhich suggests functional equivalence or similarity. As a non-limitingexample, FGF-8 is found in fish, birds and mammals and each of theseFGF-8 ligands have over 80% amino acid sequence identity. As anothernon-limiting example, FGF-18 is found in fish, birds and mammals andeach of these FGF-18 ligands have over 70% amino acid sequence identity.Crystallographic studies have revealed that all FGFs are structurallyorganized into β-trefoil domain. The amino acid sequences comprising theβ-trefoil domains found in various FGF ligands that bind FGFR3 are shownin Table 7.

TABLE 7 β-trefoil Domains of FGFs Amino Acid Sequence Region ofCarbohydrate Binding Moieties β4/β5 β8/β9 Ligand SEQ ID NO: α-foldβ-hairpin turn β-fold β-hairpin turn γ-fold FGF-1 35 26-64 65-67 68-105106-108 109-155 FGF-2 36 29-67 68-70 71-111 112-114 115-155 FGF-4 37 83-121 122-124 125-162  163-165 166-206 FGF-8 38 43-80 81-83 84-123124-126 127-172 FGF-9 39  63-100 101-103 104-144  145-147 148-196 FGF-1740 55-91 92-94 95-134 135-137 138-183 FGF-18 41 54-91 92-94 95-134135-137 138-183

As used herein, the term “Fibroblast Growth Factor” is synonymous with“FGF” and means a polypeptide with selective binding activity, such as,e.g., a binding affinity or a binding specificity, for a receptor,including endogenous Clostridial toxin receptors such as, e.g., areceptor comprising FGFR3. It is envisioned that both naturallyoccurring FGFs as well as non-naturally occurring FGFs can be used as abinding domain. Any of a variety of sequence alignment methods can beused to determine percent identity of a non-naturally occurring FGFrelative to a naturally-occurring FGF, including, without limitation,global methods, local methods and hybrid methods, such as, e.g., segmentapproach methods. Protocols to determine percent identity are routineprocedures within the scope of one skilled in the art and from theteaching herein.

Approaches well known to one skilled in the art on how to modify a FGFin order to increase its binding activity for an endogenous Clostridialtoxin receptor present on a naturally-occurring Clostridial toxin targetcell. As described above, one approach involves identifying amino acidsusing computational protein design algorithms; changingspecifically-identified amino acids using, without limitation,site-directed mutagenesis, oligonucleotide-directed mutagenesis andsite-specific mutagenesis; and testing the binding activity ofmultivalent Clostridial toxins comprising a modified FGF with enhancedbinding activity using, e.g., heterogeneous assays, homogeneous assaysand non-separating homogeneous assays. It is further envisioned that thebinding activity of a multivalent Clostridial toxin disclosed in thepresent specification can be determined by affinity chromatography usingimmobilized receptors and interfacial optical assays. In anotherapproach described above, a binding activity of a modified FGF for anaturally-occurring Clostridial toxin receptor present on anaturally-occurring Clostridial toxin target cell can be achieved usingdirected-evolution methods.

A FGF includes, without limitation, naturally occurring FGF variants,such as, e.g., FGF isoforms and FGF subtypes; non-naturally occurringFGF variants, such as, e.g., conservative FGF variants, non-conservativeFGF variants, FGF chimerics, active FGF fragments thereof, or anycombination thereof.

As used herein, the term “FGF variant,” whether naturally-occurring ornon-naturally-occurring, means a FGF that has at least one amino acidchange from the corresponding region of the disclosed referencesequences (see Table 7) and can be described in percent identity to thecorresponding region of that reference sequence. Unless expresslyindicated, all FGF variants disclosed in the present specification arecapable of executing the cell binding step of the intoxication process.

It is recognized by those of skill in the art that there can benaturally occurring FGF variants that differ somewhat in their aminoacid sequence, and also in the nucleic acids encoding these proteins.For example, there are presently at least four FGF-8 variants, FGF-8A,FGF-8A, FGF-8B, FGF-8E and FGF-8F, with specific FGF-8 variants showingvarious degrees of amino acid divergence when compared to another FGF-8variant. As another example, there are presently at least two FGF-2variants, FGF-2-1 and FGF-2-1, with the FGF-2-1 variant showing an aminoacid divergence when compared to the FGF-2-2 variant. As used herein,the term “naturally occurring FGF variant” means any FGF produced by anaturally-occurring process, including, without limitation, FGF isoformsproduced from alternatively-spliced transcripts, FGF isoforms producedby spontaneous mutation and FGF subtypes. A naturally occurring FGFvariant can function in substantially the same manner as the referenceFGF on which the naturally occurring FGF variant is based, and can besubstituted for the reference FGF in any aspect of the presentinvention. A naturally occurring FGF variant may substitute one or moreamino acids, two or more amino acids, three or more amino acids, four ormore amino acids, five or more amino acids, ten or more amino acids, 20or more amino acids, 30 or more amino acids, 40 or more amino acids, or50 or more amino acids from the reference FGF on which the naturallyoccurring FGF variant is based. A naturally occurring FGF variant canalso substitute at least 10 contiguous amino acids, at least 15contiguous amino acids, at least 20 contiguous amino acids, or at least25 contiguous amino acids from the reference FGF on which the naturallyoccurring FGF variant is based, that possess at least 50% amino acididentity, 65% amino acid identity, 75% amino acid identity, 85% aminoacid identity or 95% amino acid identity to the reference FGF on whichthe naturally occurring FGF variant is based.

A non-limiting examples of a naturally occurring FGF variant is a FGFisoform such as, e.g., a FGF-1 isoform, a FGF-2 isoform, a FGF-4isoform, a FGF-8 isoform, a FGF-9 isoform, a FGF-17 isoform and a FGF-18isoform. A FGF isoform can function in substantially the same manner asthe reference FGF on which the FGF isoform is based, and can besubstituted for the reference FGF in any aspect of the presentinvention.

Another non-limiting examples of a naturally occurring FGF variant is aFGF subtype such as, e.g., a FGF-1 subtype, a FGF-2 subtype, a FGF-4subtype, a FGF-8 subtype, a FGF-9 subtype, a FGF-17 subtype and a FGF-18subtype. A FGF subtype can function in substantially the same manner asthe reference FGF on which the FGF subtype is based, and can besubstituted for the reference FGF in any aspect of the presentinvention.

As used herein, the term “non-naturally occurring FGF variant” means anyFGF produced with the aid of human manipulation, including, withoutlimitation, FGFs produced by genetic engineering using randommutagenesis or rational design and FGFs produced by chemical synthesis.Non-limiting examples of non-naturally occurring FGF variants include,e.g., conservative FGF variants, non-conservative FGF variants, FGFchimeric variants and active FGF fragments.

As used herein, the term “conservative FGF variant” means a FGF that hasat least one amino acid substituted by another amino acid or an aminoacid analog that has at least one property similar to that of theoriginal amino acid from the reference FGF sequence (see Table 7).Examples of properties include, without limitation, similar size,topography, charge, hydrophobicity, hydrophilicity, lipophilicity,covalent-bonding capacity, hydrogen-bonding capacity, a physicochemicalproperty, of the like, or any combination thereof. A conservative FGFvariant can function in substantially the same manner as the referenceFGF on which the conservative FGF variant is based, and can besubstituted for the reference FGF in any aspect of the presentinvention. A conservative FGF variant may substitute one or more aminoacids, two or more amino acids, three or more amino acids, four or moreamino acids, five or more amino acids, ten or more amino acids, 20 ormore amino acids, 30 or more amino acids, 40 or more amino acids or 50or more amino acids from the reference FGF on which the conservative FGFvariant is based. A conservative FGF variant can also substitute atleast 10 contiguous amino acids, at least 15 contiguous amino acids, atleast 20 contiguous amino acids, or at least 25 contiguous amino acidsfrom the reference FGF on which the conservative FGF variant is based,that possess at least 50% amino acid identity, 65% amino acid identity,75% amino acid identity, 85% amino acid identity or 95% amino acididentity to the reference FGF on which the conservative FGF variant isbased. Non-limiting examples of a conservative FGF variant include,e.g., a conservative FGF-1 variant, a conservative FGF-2 variant, aconservative FGF-4 variant, a conservative FGF-8 variant, a conservativeFGF-9 variant, a conservative FGF-17 variant and a conservative FGF-18variant.

As used herein, the term “non-conservative FGF variant” means a FGF inwhich 1) at least one amino acid is deleted from the reference FGF onwhich the non-conservative FGF variant is based; 2) at least one aminoacid added to the reference FGF on which the non-conservative FGF isbased; or 3) at least one amino acid is substituted by another aminoacid or an amino acid analog that does not share any property similar tothat of the original amino acid from the reference FGF sequence (seeTable 7). A non-conservative FGF variant can function in substantiallythe same manner as the reference FGF on which the non-conservative FGFvariant is based, and can be substituted for the reference FGF in anyaspect of the present invention. A non-conservative FGF variant candelete one or more amino acids, two or more amino acids, three or moreamino acids, four or more amino acids, five or more amino acids, and tenor more amino acids from the reference FGF on which the non-conservativeFGF variant is based. A non-conservative FGF variant can add one or moreamino acids, two or more amino acids, three or more amino acids, four ormore amino acids, five or more amino acids, and ten or more amino acidsto the reference FGF on which the non-conservative FGF variant is based.A non-conservative FGF variant may substitute one or more amino acids,two or more amino acids, three or more amino acids, four or more aminoacids, five or more amino acids, ten or more amino acids, 20 or moreamino acids, 30 or more amino acids, 40 or more amino acids or 50 ormore amino acids from the reference FGF on which the non-conservativeFGF variant is based. A non-conservative FGF variant can also substituteat least 10 contiguous amino acids, at least 15 contiguous amino acids,at least 20 contiguous amino acids, or at least 25 contiguous aminoacids from the reference FGF on which the non-conservative FGF variantis based, that possess at least 50% amino acid identity, 65% amino acididentity, 75% amino acid identity, 85% amino acid identity or 95% aminoacid identity to the reference FGF on which the non-conservative FGFvariant is based. Non-limiting examples of a non-conservative FGFvariant include, e.g., a non-conservative FGF-1 variant, anon-conservative FGF-2 variant, a non-conservative FGF-4 variant, anon-conservative FGF-8 variant, a non-conservative FGF-9 variant, anon-conservative FGF-17 variant and a non-conservative FGF-18 variant.

As used herein, the term “FGF chimeric” means a polypeptide comprisingat least a portion of a FGF and at least a portion of at least one otherpolypeptide to form an enhanced targeting domain with at least oneproperty different from the reference FGF (see Table 7), with theproviso that this FGF chimeric can specifically bind to an endogenousClostridial toxin receptor present in a Clostridial toxin target cell,and thus participate in executing the overall cellular mechanism wherebya Clostridial toxin proteolytically cleaves a substrate.

Thus, in an embodiment, a binding domain comprises a FGF. In aspects ofthis embodiment, a FGF comprises a β-trefoil domain derived from a FGF.In an aspect of this embodiment, a β-trefoil domain derived from a FGFcomprises, e.g., a β-trefoil domain derived from a FGF-1, a β-trefoildomain derived from a FGF-2, a β-trefoil domain derived from FGF-4, aβ-trefoil domain derived from a FGF-8, a β-trefoil domain derived from aFGF-9, a β-trefoil domain derived from a FGF-17 or a β-trefoil domainderived from a FGF-18. In another aspect of this embodiment, a β-trefoildomain derived from a FGF comprises a α-fold motif of a β-trefoil domainof a FGF, a β-fold motif of a β-trefoil domain of a FGF or a γ-foldmotif of a β-trefoil domain of a FGF.

In another embodiment, a binding domain comprises a β-trefoil domainderived a FGF-1 of SEQ ID NO: 35. In another embodiment, a bindingdomain comprising a β-trefoil domain derived from a FGF-1 comprisesamino acids 26-155 of SEQ ID NO: 35. In another aspect of thisembodiment, a binding domain comprising a β-trefoil domain derived froma FGF-1 comprises an α-fold motif of a β-trefoil domain of a FGF-1, aβ-fold motif of a β-trefoil domain of a FGF-1 or a γ-fold motif of aβ-trefoil domain of a FGF-1 of SEQ ID NO: 35. In another aspect of thisembodiment, a β-trefoil domain derived from a FGF-1 comprises aminoacids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO:35.

In other aspects of this embodiment, a binding domain comprising a FGF-1β-trefoil domain comprises a polypeptide having, e.g., at least 70%amino acid identity with amino acids 26-64, amino acids 68-105, or aminoacids 109-155 of SEQ ID NO: 35, at least 75% amino acid identity withamino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ IDNO: 35, at least 80% amino acid identity with amino acids 26-64, aminoacids 68-105, or amino acids 109-155 of SEQ ID NO: 35, at least 85%amino acid identity with amino acids 26-64, amino acids 68-105, or aminoacids 109-155 of SEQ ID NO: 35, at least 90% amino acid identity withamino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ IDNO: 35 or at least 95% amino acid identity with amino acids 26-64, aminoacids 68-105, or amino acids 109-155 of SEQ ID NO: 35. In yet otheraspects of this embodiment, a binding domain comprising a FGF-1β-trefoil domain comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 26-64, amino acids 68-105, or amino acids109-155 of SEQ ID NO: 35, at most 75% amino acid identity with aminoacids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO:35, at most 80% amino acid identity with amino acids 26-64, amino acids68-105, or amino acids 109-155 of SEQ ID NO: 35, at most 85% amino acididentity with amino acids 26-64, amino acids 68-105, or amino acids109-155 of SEQ ID NO: 35, at most 90% amino acid identity with aminoacids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35or at most 95% amino acid identity with amino acids 26-64, amino acids68-105, or amino acids 109-155 of SEQ ID NO: 35.

In other aspects of this embodiment, a binding domain comprising a FGF-1β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 26-64, amino acids68-105, or amino acids 109-155 of SEQ ID NO: 35. In other aspects ofthis embodiment, a binding domain comprising a FGF-1 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 26-64, amino acids 68-105, oramino acids 109-155 of SEQ ID NO: 35. In yet other aspects of thisembodiment, a binding domain comprising a FGF-1 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 26-64, amino acids 68-105, or aminoacids 109-155 of SEQ ID NO: 35. In other aspects of this embodiment, abinding domain comprising a FGF-1 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 26-64, amino acids 68-105, or amino acids109-155 of SEQ ID NO: 35. In still other aspects of this embodiment, abinding domain comprising a FGF-1 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 26-64, amino acids 68-105, or amino acids109-155 of SEQ ID NO: 35. In other aspects of this embodiment, a bindingdomain comprising a FGF-1 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO:35.

In other aspects of this embodiment, a binding domain comprising a FGF-1β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid substitutions relative to amino acids 26-64, amino acids 68-105, oramino acids 109-155 of SEQ ID NO: 35. In other aspects of thisembodiment, a binding domain comprising a FGF-1 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 26-64, amino acids 68-105, oramino acids 109-155 of SEQ ID NO: 35. In yet other aspects of thisembodiment, a binding domain comprising a FGF-1 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 26-64, amino acids 68-105, or amino acids109-155 of SEQ ID NO: 35. In other aspects of this embodiment, a bindingdomain comprising a FGF-1 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 35. Instill other aspects of this embodiment, a binding domain comprising aFGF-1 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 26-64, aminoacids 68-105, or amino acids 109-155 of SEQ ID NO: 35. In other aspectsof this embodiment, a binding domain comprising a FGF-1 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 26-64, amino acids 68-105, or amino acids109-155 of SEQ ID NO: 35.

In another embodiment, a binding domain comprising a FGF-1 β-trefoildomain comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-1or a β8/β9 hairpin turn of a β-trefoil domain of a FGF-1 of SEQ ID NO:35. In another aspect of this embodiment, a binding domain comprising aFGF-1 β-trefoil domain comprises amino acids 65-67 or amino acids106-108 of SEQ ID NO: 35.

In other aspects of this embodiment, a binding domain comprising a FGF-1β-trefoil domain comprises a polypeptide having, e.g., at least 70%amino acid identity with amino acids 65-67 or amino acids 106-108 of SEQID NO: 35, at least 75% amino acid identity with amino acids 65-67 oramino acids 106-108 of SEQ ID NO: 35, at least 80% amino acid identitywith amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35, at least85% amino acid identity with amino acids 65-67 or amino acids 106-108 ofSEQ ID NO: 35, at least 90% amino acid identity with amino acids 65-67or amino acids 106-108 of SEQ ID NO: 35 or at least 95% amino acididentity with amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35.In yet other aspects of this embodiment, a binding domain comprising aFGF-1 β-trefoil domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 65-67 or amino acids 106-108 of SEQID NO: 35, at most 75% amino acid identity with amino acids 65-67 oramino acids 106-108 of SEQ ID NO: 35, at most 80% amino acid identitywith amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35, at most85% amino acid identity with amino acids 65-67 or amino acids 106-108 ofSEQ ID NO: 35, at most 90% amino acid identity with amino acids 65-67 oramino acids 106-108 of SEQ ID NO: 35 or at most 95% amino acid identitywith amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35.

In other aspects of this embodiment, a binding domain comprising a FGF-1β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four non-contiguous amino acid substitutions relative to aminoacids 65-67 or amino acids 106-108 of SEQ ID NO: 35. In other aspects ofthis embodiment, a binding domain comprising a FGF-1 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 65-67 oramino acids 106-108 of SEQ ID NO: 35. In other aspects of thisembodiment, a non-contiguous amino acid substitution of any amino acidfrom amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35 can bereplaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35 can bereplaced with phenylalanine. In yet other aspects of this embodiment, abinding domain comprising a FGF-1 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 65-67 or amino acids106-108 of SEQ ID NO: 35. In other aspects of this embodiment, a bindingdomain comprising a FGF-1 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino aciddeletions relative to amino acids 65-67 or amino acids 106-108 of SEQ IDNO: 35. In still other aspects of this embodiment, a binding domaincomprising a FGF-1 β-trefoil domain comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino acidadditions relative to amino acids 65-67 or amino acids 106-108 of SEQ IDNO: 35. In other aspects of this embodiment, a binding domain comprisinga FGF-1 β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three or four non-contiguous amino acid additions relative toamino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35.

In other aspects of this embodiment, a binding domain comprising a FGF-1β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid substitutions relative to aminoacids 65-67 or amino acids 106-108 of SEQ ID NO: 35. In other aspects ofthis embodiment, a binding domain comprising a FGF-1 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 65-67 oramino acids 106-108 of SEQ ID NO: 35. In other aspects of thisembodiment, contiguous amino acid substitutions of amino acids fromamino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35 can bereplaced with glycine. In other aspects of this embodiment, contiguousamino acid substitutions of hydrophobic amino acids from amino acids65-67 or amino acids 106-108 of SEQ ID NO: 35 can be replaced withphenylalanine. In yet other aspects of this embodiment, a binding domaincomprising a FGF-1 β-trefoil domain comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 35.In other aspects of this embodiment, a binding domain comprising a FGF-1β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four contiguous amino acid deletions relative to aminoacids 65-67 or amino acids 106-108 of SEQ ID NO: 35. In still otheraspects of this embodiment, a binding domain comprising a FGF-1β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids65-67 or amino acids 106-108 of SEQ ID NO: 35. In other aspects of thisembodiment, a binding domain comprising a FGF-1 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 65-67 or aminoacids 106-108 of SEQ ID NO: 35.

In another embodiment, a binding domain comprises a β-trefoil domainderived a FGF-2 of SEQ ID NO: 36. In another embodiment, a bindingdomain comprising a β-trefoil domain derived from a FGF-2 comprisesamino acids 29-155 of SEQ ID NO: 36. In another aspect of thisembodiment, a binding domain comprising a β-trefoil domain derived froma FGF-2 comprises an α-fold motif of a β-trefoil domain of a FGF-2, aβ-fold motif of a β-trefoil domain of a FGF-2 or a γ-fold motif of aβ-trefoil domain of a FGF-2 of SEQ ID NO: 36. In another aspect of thisembodiment, a β-trefoil domain derived from a FGF-2 comprises aminoacids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO:36.

In other aspects of this embodiment, a binding domain comprising a FGF-2β-trefoil domain comprises a polypeptide having, e.g., at least 70%amino acid identity with amino acids 29-67, amino acids 71-111, or aminoacids 115-155 of SEQ ID NO: 36, at least 75% amino acid identity withamino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ IDNO: 36, at least 80% amino acid identity with amino acids 29-67, aminoacids 71-111, or amino acids 115-155 of SEQ ID NO: 36, at least 85%amino acid identity with amino acids 29-67, amino acids 71-111, or aminoacids 115-155 of SEQ ID NO: 36, at least 90% amino acid identity withamino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ IDNO: 36 or at least 95% amino acid identity with amino acids 29-67, aminoacids 71-111, or amino acids 115-155 of SEQ ID NO: 36. In yet otheraspects of this embodiment, a binding domain comprising a FGF-2β-trefoil domain comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 29-67, amino acids 71-111, or amino acids115-155 of SEQ ID NO: 36, at most 75% amino acid identity with aminoacids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO:36, at most 80% amino acid identity with amino acids 29-67, amino acids71-111, or amino acids 115-155 of SEQ ID NO: 36, at most 85% amino acididentity with amino acids 29-67, amino acids 71-111, or amino acids115-155 of SEQ ID NO: 36, at most 90% amino acid identity with aminoacids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36or at most 95% amino acid identity with amino acids 29-67, amino acids71-111, or amino acids 115-155 of SEQ ID NO: 36.

In other aspects of this embodiment, a binding domain comprising a FGF-2β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 29-67, amino acids71-111, or amino acids 115-155 of SEQ ID NO: 36. In other aspects ofthis embodiment, a binding domain comprising a FGF-2 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 29-67, amino acids 71-111, oramino acids 115-155 of SEQ ID NO: 36. In yet other aspects of thisembodiment, a binding domain comprising a FGF-2 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 29-67, amino acids 71-111, or aminoacids 115-155 of SEQ ID NO: 36. In other aspects of this embodiment, abinding domain comprising a FGF-2 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 29-67, amino acids 71-111, or amino acids115-155 of SEQ ID NO: 36. In still other aspects of this embodiment, abinding domain comprising a FGF-2 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 29-67, amino acids 71-111, or amino acids115-155 of SEQ ID NO: 36. In other aspects of this embodiment, a bindingdomain comprising a FGF-2 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO:36.

In other aspects of this embodiment, a binding domain comprising a FGF-2β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid substitutions relative to amino acids 29-67, amino acids 71-111, oramino acids 115-155 of SEQ ID NO: 36. In other aspects of thisembodiment, a binding domain comprising a FGF-2 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 29-67, amino acids 71-111, oramino acids 115-155 of SEQ ID NO: 36. In yet other aspects of thisembodiment, a binding domain comprising a FGF-2 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 29-67, amino acids 71-111, or amino acids115-155 of SEQ ID NO: 36. In other aspects of this embodiment, a bindingdomain comprising a FGF-2 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 36. Instill other aspects of this embodiment, a binding domain comprising aFGF-2 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 29-67, aminoacids 71-111, or amino acids 115-155 of SEQ ID NO: 36. In other aspectsof this embodiment, a binding domain comprising a FGF-2 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 29-67, amino acids 71-111, or amino acids115-155 of SEQ ID NO: 36.

In another embodiment, a binding domain comprising a FGF-2 β-trefoildomain comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-2or a β8/β9 hairpin turn of a β-trefoil domain of a FGF-2 of SEQ ID NO:35. In another aspect of this embodiment, a binding domain comprising aFGF-2 β-trefoil domain comprises amino acids 68-70 or amino acids112-114 of SEQ ID NO: 36.

In other aspects of this embodiment, a binding domain comprising a FGF-2β-trefoil domain comprises a polypeptide having, e.g., at least 70%amino acid identity with amino acids 68-70 or amino acids 112-114 of SEQID NO: 36, at least 75% amino acid identity with amino acids 68-70 oramino acids 112-114 of SEQ ID NO: 36, at least 80% amino acid identitywith amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36, at least85% amino acid identity with amino acids 68-70 or amino acids 112-114 ofSEQ ID NO: 36, at least 90% amino acid identity with amino acids 68-70or amino acids 112-114 of SEQ ID NO: 36 or at least 95% amino acididentity with amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36.In yet other aspects of this embodiment, a binding domain comprising aFGF-2 β-trefoil domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 68-70 or amino acids 112-114 of SEQID NO: 36, at most 75% amino acid identity with amino acids 68-70 oramino acids 112-114 of SEQ ID NO: 36, at most 80% amino acid identitywith amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36, at most85% amino acid identity with amino acids 68-70 or amino acids 112-114 ofSEQ ID NO: 36, at most 90% amino acid identity with amino acids 68-70 oramino acids 112-114 of SEQ ID NO: 36 or at most 95% amino acid identitywith amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36.

In other aspects of this embodiment, a binding domain comprising a FGF-2β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four non-contiguous amino acid substitutions relative to aminoacids 68-70 or amino acids 112-114 of SEQ ID NO: 36. In other aspects ofthis embodiment, a binding domain comprising a FGF-2 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 68-70 oramino acids 112-114 of SEQ ID NO: 36. In other aspects of thisembodiment, a non-contiguous amino acid substitution of any amino acidfrom amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36 can bereplaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36 can bereplaced with phenylalanine. In yet other aspects of this embodiment, abinding domain comprising a FGF-2 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 68-70 or amino acids112-114 of SEQ ID NO: 36. In other aspects of this embodiment, a bindingdomain comprising a FGF-2 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino aciddeletions relative to amino acids 68-70 or amino acids 112-114 of SEQ IDNO: 36. In still other aspects of this embodiment, a binding domaincomprising a FGF-2 β-trefoil domain comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino acidadditions relative to amino acids 68-70 or amino acids 112-114 of SEQ IDNO: 36. In other aspects of this embodiment, a binding domain comprisinga FGF-2 β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three or four non-contiguous amino acid additions relative toamino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36.

In other aspects of this embodiment, a binding domain comprising a FGF-2β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid substitutions relative to aminoacids 68-70 or amino acids 112-114 of SEQ ID NO: 36. In other aspects ofthis embodiment, a binding domain comprising a FGF-2 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 68-70 oramino acids 112-114 of SEQ ID NO: 36. In other aspects of thisembodiment, contiguous amino acid substitutions of amino acids fromamino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36 can bereplaced with glycine. In other aspects of this embodiment, contiguousamino acid substitutions of hydrophobic amino acids from amino acids68-70 or amino acids 112-114 of SEQ ID NO: 36 can be replaced withphenylalanine. In yet other aspects of this embodiment, a binding domaincomprising a FGF-2 β-trefoil domain comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 36.In other aspects of this embodiment, a binding domain comprising a FGF-2β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four contiguous amino acid deletions relative to aminoacids 68-70 or amino acids 112-114 of SEQ ID NO: 36. In still otheraspects of this embodiment, a binding domain comprising a FGF-2β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids68-70 or amino acids 112-114 of SEQ ID NO: 36. In other aspects of thisembodiment, a binding domain comprising a FGF-2 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 68-70 or aminoacids 112-114 of SEQ ID NO: 36.

In another embodiment, a binding domain comprises a β-trefoil domainderived a FGF-4 of SEQ ID NO: 37. In another embodiment, a bindingdomain comprising a β-trefoil domain derived from a FGF-4 comprisesamino acids 83-206 of SEQ ID NO: 37. In another aspect of thisembodiment, a binding domain comprising a β-trefoil domain derived froma FGF-4 comprises an α-fold motif of a β-trefoil domain of a FGF-4, aβ-fold motif of a β-trefoil domain of a FGF-4 or a γ-fold motif of aβ-trefoil domain of a FGF-4 of SEQ ID NO: 37. In another aspect of thisembodiment, a β-trefoil domain derived from a FGF-4 comprises aminoacids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO:37.

In other aspects of this embodiment, a binding domain comprising a FGF-4β-trefoil domain comprises a polypeptide having, e.g., at least 70%amino acid identity with amino acids 83-121, amino acids 125-162, oramino acids 166-206 of SEQ ID NO: 37, at least 75% amino acid identitywith amino acids 83-121, amino acids 125-162, or amino acids 166-206 ofSEQ ID NO: 37, at least 80% amino acid identity with amino acids 83-121,amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37, at least85% amino acid identity with amino acids 83-121, amino acids 125-162, oramino acids 166-206 of SEQ ID NO: 37, at least 90% amino acid identitywith amino acids 83-121, amino acids 125-162, or amino acids 166-206 ofSEQ ID NO: 37 or at least 95% amino acid identity with amino acids83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37. Inyet other aspects of this embodiment, a binding domain comprising aFGF-4 β-trefoil domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 83-121, amino acids 125-162, oramino acids 166-206 of SEQ ID NO: 37, at most 75% amino acid identitywith amino acids 83-121, amino acids 125-162, or amino acids 166-206 ofSEQ ID NO: 37, at most 80% amino acid identity with amino acids 83-121,amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37, at most85% amino acid identity with amino acids 83-121, amino acids 125-162, oramino acids 166-206 of SEQ ID NO: 37, at most 90% amino acid identitywith amino acids 83-121, amino acids 125-162, or amino acids 166-206 ofSEQ ID NO: 37 or at most 95% amino acid identity with amino acids83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37.

In other aspects of this embodiment, a binding domain comprising a FGF-4β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 83-121, amino acids125-162, or amino acids 166-206 of SEQ ID NO: 37. In other aspects ofthis embodiment, a binding domain comprising a FGF-4 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 83-121, amino acids 125-162, oramino acids 166-206 of SEQ ID NO: 37. In yet other aspects of thisembodiment, a binding domain comprising a FGF-4 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 83-121, amino acids 125-162, or aminoacids 166-206 of SEQ ID NO: 37. In other aspects of this embodiment, abinding domain comprising a FGF-4 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 83-121, amino acids 125-162, or amino acids166-206 of SEQ ID NO: 37. In still other aspects of this embodiment, abinding domain comprising a FGF-4 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 83-121, amino acids 125-162, or amino acids166-206 of SEQ ID NO: 37. In other aspects of this embodiment, a bindingdomain comprising a FGF-4 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO:37.

In other aspects of this embodiment, a binding domain comprising a FGF-4β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid substitutions relative to amino acids 83-121, amino acids 125-162,or amino acids 166-206 of SEQ ID NO: 37. In other aspects of thisembodiment, a binding domain comprising a FGF-4 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 83-121, amino acids 125-162, oramino acids 166-206 of SEQ ID NO: 37. In yet other aspects of thisembodiment, a binding domain comprising a FGF-4 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 83-121, amino acids 125-162, or amino acids166-206 of SEQ ID NO: 37. In other aspects of this embodiment, a bindingdomain comprising a FGF-4 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 37. Instill other aspects of this embodiment, a binding domain comprising aFGF-4 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 83-121, aminoacids 125-162, or amino acids 166-206 of SEQ ID NO: 37. In other aspectsof this embodiment, a binding domain comprising a FGF-4 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 83-121, amino acids 125-162, or amino acids166-206 of SEQ ID NO: 37.

In another embodiment, a binding domain comprising a FGF-4 β-trefoildomain comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-4or a β8/β9 hairpin turn of a β-trefoil domain of a FGF-4 of SEQ ID NO:35. In another aspect of this embodiment, a binding domain comprising aFGF-4 β-trefoil domain comprises amino acids 122-124 or amino acids163-165 of SEQ ID NO: 37.

In other aspects of this embodiment, a binding domain comprising a FGF-4β-trefoil domain comprises a polypeptide having, e.g., at least 70%amino acid identity with amino acids 122-124 or amino acids 163-165 ofSEQ ID NO: 37, at least 75% amino acid identity with amino acids 122-124or amino acids 163-165 of SEQ ID NO: 37, at least 80% amino acididentity with amino acids 122-124 or amino acids 163-165 of SEQ ID NO:37, at least 85% amino acid identity with amino acids 122-124 or aminoacids 163-165 of SEQ ID NO: 37, at least 90% amino acid identity withamino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37 or at least95% amino acid identity with amino acids 122-124 or amino acids 163-165of SEQ ID NO: 37. In yet other aspects of this embodiment, a bindingdomain comprising a FGF-4 β-trefoil domain comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 122-124or amino acids 163-165 of SEQ ID NO: 37, at most 75% amino acid identitywith amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37, atmost 80% amino acid identity with amino acids 122-124 or amino acids163-165 of SEQ ID NO: 37, at most 85% amino acid identity with aminoacids 122-124 or amino acids 163-165 of SEQ ID NO: 37, at most 90% aminoacid identity with amino acids 122-124 or amino acids 163-165 of SEQ IDNO: 37 or at most 95% amino acid identity with amino acids 122-124 oramino acids 163-165 of SEQ ID NO: 37.

In other aspects of this embodiment, a binding domain comprising a FGF-4β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four non-contiguous amino acid substitutions relative to aminoacids 122-124 or amino acids 163-165 of SEQ ID NO: 37. In other aspectsof this embodiment, a binding domain comprising a FGF-4 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 122-124or amino acids 163-165 of SEQ ID NO: 37. In other aspects of thisembodiment, a non-contiguous amino acid substitution of any amino acidfrom amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37 can bereplaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37 can bereplaced with phenylalanine. In yet other aspects of this embodiment, abinding domain comprising a FGF-4 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 122-124 or amino acids163-165 of SEQ ID NO: 37. In other aspects of this embodiment, a bindingdomain comprising a FGF-4 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino aciddeletions relative to amino acids 122-124 or amino acids 163-165 of SEQID NO: 37. In still other aspects of this embodiment, a binding domaincomprising a FGF-4 β-trefoil domain comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino acidadditions relative to amino acids 122-124 or amino acids 163-165 of SEQID NO: 37. In other aspects of this embodiment, a binding domaincomprising a FGF-4 β-trefoil domain comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 122-124 or amino acids 163-165 of SEQID NO: 37.

In other aspects of this embodiment, a binding domain comprising a FGF-4β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid substitutions relative to aminoacids 122-124 or amino acids 163-165 of SEQ ID NO: 37. In other aspectsof this embodiment, a binding domain comprising a FGF-4 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 122-124 oramino acids 163-165 of SEQ ID NO: 37. In other aspects of thisembodiment, contiguous amino acid substitutions of amino acids fromamino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37 can bereplaced with glycine. In other aspects of this embodiment, contiguousamino acid substitutions of hydrophobic amino acids from amino acids122-124 or amino acids 163-165 of SEQ ID NO: 37 can be replaced withphenylalanine. In yet other aspects of this embodiment, a binding domaincomprising a FGF-4 β-trefoil domain comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 37.In other aspects of this embodiment, a binding domain comprising a FGF-4β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four contiguous amino acid deletions relative to aminoacids 122-124 or amino acids 163-165 of SEQ ID NO: 37. In still otheraspects of this embodiment, a binding domain comprising a FGF-4β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids122-124 or amino acids 163-165 of SEQ ID NO: 37. In other aspects ofthis embodiment, a binding domain comprising a FGF-4 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 122-124 or aminoacids 163-165 of SEQ ID NO: 37.

In another embodiment, a binding domain comprises a β-trefoil domainderived a FGF-8 of SEQ ID NO: 38. In another embodiment, a bindingdomain comprising a β-trefoil domain derived from a FGF-8 comprisesamino acids 43-172 of SEQ ID NO: 38. In another aspect of thisembodiment, a binding domain comprising a β-trefoil domain derived froma FGF-8 comprises an α-fold motif of a β-trefoil domain of a FGF-8, aβ-fold motif of a β-trefoil domain of a FGF-8 or a γ-fold motif of aβ-trefoil domain of a FGF-8 of SEQ ID NO: 38. In another aspect of thisembodiment, a β-trefoil domain derived from a FGF-8 comprises aminoacids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO:38.

In other aspects of this embodiment, a binding domain comprising a FGF-8β-trefoil domain comprises a polypeptide having, e.g., at least 70%amino acid identity with amino acids 43-80, amino acids 84-123, or aminoacids 127-172 of SEQ ID NO: 38, at least 75% amino acid identity withamino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ IDNO: 38, at least 80% amino acid identity with amino acids 43-80, aminoacids 84-123, or amino acids 127-172 of SEQ ID NO: 38, at least 85%amino acid identity with amino acids 43-80, amino acids 84-123, or aminoacids 127-172 of SEQ ID NO: 38, at least 90% amino acid identity withamino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ IDNO: 38 or at least 95% amino acid identity with amino acids 43-80, aminoacids 84-123, or amino acids 127-172 of SEQ ID NO: 38. In yet otheraspects of this embodiment, a binding domain comprising a FGF-8β-trefoil domain comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 43-80, amino acids 84-123, or amino acids127-172 of SEQ ID NO: 38, at most 75% amino acid identity with aminoacids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO:38, at most 80% amino acid identity with amino acids 43-80, amino acids84-123, or amino acids 127-172 of SEQ ID NO: 38, at most 85% amino acididentity with amino acids 43-80, amino acids 84-123, or amino acids127-172 of SEQ ID NO: 38, at most 90% amino acid identity with aminoacids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38or at most 95% amino acid identity with amino acids 43-80, amino acids84-123, or amino acids 127-172 of SEQ ID NO: 38.

In other aspects of this embodiment, a binding domain comprising a FGF-8β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 43-80, amino acids84-123, or amino acids 127-172 of SEQ ID NO: 38. In other aspects ofthis embodiment, a binding domain comprising a FGF-8 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 43-80, amino acids 84-123, oramino acids 127-172 of SEQ ID NO: 38. In yet other aspects of thisembodiment, a binding domain comprising a FGF-8 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 43-80, amino acids 84-123, or aminoacids 127-172 of SEQ ID NO: 38. In other aspects of this embodiment, abinding domain comprising a FGF-8 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 43-80, amino acids 84-123, or amino acids127-172 of SEQ ID NO: 38. In still other aspects of this embodiment, abinding domain comprising a FGF-8 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 43-80, amino acids 84-123, or amino acids127-172 of SEQ ID NO: 38. In other aspects of this embodiment, a bindingdomain comprising a FGF-8 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO:38.

In other aspects of this embodiment, a binding domain comprising a FGF-8β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid substitutions relative to amino acids 43-80, amino acids 84-123, oramino acids 127-172 of SEQ ID NO: 38. In other aspects of thisembodiment, a binding domain comprising a FGF-8 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 43-80, amino acids 84-123, oramino acids 127-172 of SEQ ID NO: 38. In yet other aspects of thisembodiment, a binding domain comprising a FGF-8 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 43-80, amino acids 84-123, or amino acids127-172 of SEQ ID NO: 38. In other aspects of this embodiment, a bindingdomain comprising a FGF-8 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 38. Instill other aspects of this embodiment, a binding domain comprising aFGF-8 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 43-80, aminoacids 84-123, or amino acids 127-172 of SEQ ID NO: 38. In other aspectsof this embodiment, a binding domain comprising a FGF-8 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 43-80, amino acids 84-123, or amino acids127-172 of SEQ ID NO: 38.

In another embodiment, a binding domain comprising a FGF-8 β-trefoildomain comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-8or a β8/β9 hairpin turn of a β-trefoil domain of a FGF-8 of SEQ ID NO:35. In another aspect of this embodiment, a binding domain comprising aFGF-8 β-trefoil domain comprises amino acids 81-83 or amino acids124-126 of SEQ ID NO: 38.

In other aspects of this embodiment, a binding domain comprising a FGF-8β-trefoil domain comprises a polypeptide having, e.g., at least 70%amino acid identity with amino acids 81-83 or amino acids 124-126 of SEQID NO: 38, at least 75% amino acid identity with amino acids 81-83 oramino acids 124-126 of SEQ ID NO: 38, at least 80% amino acid identitywith amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38, at least85% amino acid identity with amino acids 81-83 or amino acids 124-126 ofSEQ ID NO: 38, at least 90% amino acid identity with amino acids 81-83or amino acids 124-126 of SEQ ID NO: 38 or at least 95% amino acididentity with amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38.In yet other aspects of this embodiment, a binding domain comprising aFGF-8 β-trefoil domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 81-83 or amino acids 124-126 of SEQID NO: 38, at most 75% amino acid identity with amino acids 81-83 oramino acids 124-126 of SEQ ID NO: 38, at most 80% amino acid identitywith amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38, at most85% amino acid identity with amino acids 81-83 or amino acids 124-126 ofSEQ ID NO: 38, at most 90% amino acid identity with amino acids 81-83 oramino acids 124-126 of SEQ ID NO: 38 or at most 95% amino acid identitywith amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38.

In other aspects of this embodiment, a binding domain comprising a FGF-8β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four non-contiguous amino acid substitutions relative to aminoacids 81-83 or amino acids 124-126 of SEQ ID NO: 38. In other aspects ofthis embodiment, a binding domain comprising a FGF-8 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 81-83 oramino acids 124-126 of SEQ ID NO: 38. In other aspects of thisembodiment, a non-contiguous amino acid substitution of any amino acidfrom amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38 can bereplaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38 can bereplaced with phenylalanine. In yet other aspects of this embodiment, abinding domain comprising a FGF-8 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 81-83 or amino acids124-126 of SEQ ID NO: 38. In other aspects of this embodiment, a bindingdomain comprising a FGF-8 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino aciddeletions relative to amino acids 81-83 or amino acids 124-126 of SEQ IDNO: 38. In still other aspects of this embodiment, a binding domaincomprising a FGF-8 β-trefoil domain comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino acidadditions relative to amino acids 81-83 or amino acids 124-126 of SEQ IDNO: 38. In other aspects of this embodiment, a binding domain comprisinga FGF-8 β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three or four non-contiguous amino acid additions relative toamino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38.

In other aspects of this embodiment, a binding domain comprising a FGF-8β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid substitutions relative to aminoacids 81-83 or amino acids 124-126 of SEQ ID NO: 38. In other aspects ofthis embodiment, a binding domain comprising a FGF-8 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 81-83 oramino acids 124-126 of SEQ ID NO: 38. In other aspects of thisembodiment, contiguous amino acid substitutions of amino acids fromamino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38 can bereplaced with glycine. In other aspects of this embodiment, contiguousamino acid substitutions of hydrophobic amino acids from amino acids81-83 or amino acids 124-126 of SEQ ID NO: 38 can be replaced withphenylalanine. In yet other aspects of this embodiment, a binding domaincomprising a FGF-8 β-trefoil domain comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 38.In other aspects of this embodiment, a binding domain comprising a FGF-8β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four contiguous amino acid deletions relative to aminoacids 81-83 or amino acids 124-126 of SEQ ID NO: 38. In still otheraspects of this embodiment, a binding domain comprising a FGF-8β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids81-83 or amino acids 124-126 of SEQ ID NO: 38. In other aspects of thisembodiment, a binding domain comprising a FGF-8 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 81-83 or aminoacids 124-126 of SEQ ID NO: 38.

In another embodiment, a binding domain comprises a β-trefoil domainderived a FGF-9 of SEQ ID NO: 39. In another embodiment, a bindingdomain comprising a β-trefoil domain derived from a FGF-9 comprisesamino acids 63-196 of SEQ ID NO: 39. In another aspect of thisembodiment, a binding domain comprising a β-trefoil domain derived froma FGF-9 comprises an α-fold motif of a β-trefoil domain of a FGF-9, aβ-fold motif of a β-trefoil domain of a FGF-9 or a γ-fold motif of aβ-trefoil domain of a FGF-9 of SEQ ID NO: 39. In another aspect of thisembodiment, a β-trefoil domain derived from a FGF-9 comprises aminoacids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO:39.

In other aspects of this embodiment, a binding domain comprising a FGF-9β-trefoil domain comprises a polypeptide having, e.g., at least 70%amino acid identity with amino acids 63-100, amino acids 104-144, oramino acids 148-196 of SEQ ID NO: 39, at least 75% amino acid identitywith amino acids 63-100, amino acids 104-144, or amino acids 148-196 ofSEQ ID NO: 39, at least 80% amino acid identity with amino acids 63-100,amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39, at least85% amino acid identity with amino acids 63-100, amino acids 104-144, oramino acids 148-196 of SEQ ID NO: 39, at least 90% amino acid identitywith amino acids 63-100, amino acids 104-144, or amino acids 148-196 ofSEQ ID NO: 39 or at least 95% amino acid identity with amino acids63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39. Inyet other aspects of this embodiment, a binding domain comprising aFGF-9 β-trefoil domain comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 63-100, amino acids 104-144, oramino acids 148-196 of SEQ ID NO: 39, at most 75% amino acid identitywith amino acids 63-100, amino acids 104-144, or amino acids 148-196 ofSEQ ID NO: 39, at most 80% amino acid identity with amino acids 63-100,amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39, at most85% amino acid identity with amino acids 63-100, amino acids 104-144, oramino acids 148-196 of SEQ ID NO: 39, at most 90% amino acid identitywith amino acids 63-100, amino acids 104-144, or amino acids 148-196 ofSEQ ID NO: 39 or at most 95% amino acid identity with amino acids63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39.

In other aspects of this embodiment, a binding domain comprising a FGF-9β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 63-100, amino acids104-144, or amino acids 148-196 of SEQ ID NO: 39. In other aspects ofthis embodiment, a binding domain comprising a FGF-9 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 63-100, amino acids 104-144, oramino acids 148-196 of SEQ ID NO: 39. In yet other aspects of thisembodiment, a binding domain comprising a FGF-9 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 63-100, amino acids 104-144, or aminoacids 148-196 of SEQ ID NO: 39. In other aspects of this embodiment, abinding domain comprising a FGF-9 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 63-100, amino acids 104-144, or amino acids148-196 of SEQ ID NO: 39. In still other aspects of this embodiment, abinding domain comprising a FGF-9 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 63-100, amino acids 104-144, or amino acids148-196 of SEQ ID NO: 39. In other aspects of this embodiment, a bindingdomain comprising a FGF-9 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO:39.

In other aspects of this embodiment, a binding domain comprising a FGF-9β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid substitutions relative to amino acids 63-100, amino acids 104-144,or amino acids 148-196 of SEQ ID NO: 39. In other aspects of thisembodiment, a binding domain comprising a FGF-9 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 63-100, amino acids 104-144, oramino acids 148-196 of SEQ ID NO: 39. In yet other aspects of thisembodiment, a binding domain comprising a FGF-9 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 63-100, amino acids 104-144, or amino acids148-196 of SEQ ID NO: 39. In other aspects of this embodiment, a bindingdomain comprising a FGF-9 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 39. Instill other aspects of this embodiment, a binding domain comprising aFGF-9 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 63-100, aminoacids 104-144, or amino acids 148-196 of SEQ ID NO: 39. In other aspectsof this embodiment, a binding domain comprising a FGF-9 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 63-100, amino acids 104-144, or amino acids148-196 of SEQ ID NO: 39.

In another embodiment, a binding domain comprising a FGF-9 β-trefoildomain comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-9or a β8/β9 hairpin turn of a β-trefoil domain of a FGF-9 of SEQ ID NO:35. In another aspect of this embodiment, a binding domain comprising aFGF-9 β-trefoil domain comprises amino acids 101-103 or amino acids145-147 of SEQ ID NO: 39.

In other aspects of this embodiment, a binding domain comprising a FGF-9β-trefoil domain comprises a polypeptide having, e.g., at least 70%amino acid identity with amino acids 101-103 or amino acids 145-147 ofSEQ ID NO: 39, at least 75% amino acid identity with amino acids 101-103or amino acids 145-147 of SEQ ID NO: 39, at least 80% amino acididentity with amino acids 101-103 or amino acids 145-147 of SEQ ID NO:39, at least 85% amino acid identity with amino acids 101-103 or aminoacids 145-147 of SEQ ID NO: 39, at least 90% amino acid identity withamino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39 or at least95% amino acid identity with amino acids 101-103 or amino acids 145-147of SEQ ID NO: 39. In yet other aspects of this embodiment, a bindingdomain comprising a FGF-9 β-trefoil domain comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 101-103or amino acids 145-147 of SEQ ID NO: 39, at most 75% amino acid identitywith amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39, atmost 80% amino acid identity with amino acids 101-103 or amino acids145-147 of SEQ ID NO: 39, at most 85% amino acid identity with aminoacids 101-103 or amino acids 145-147 of SEQ ID NO: 39, at most 90% aminoacid identity with amino acids 101-103 or amino acids 145-147 of SEQ IDNO: 39 or at most 95% amino acid identity with amino acids 101-103 oramino acids 145-147 of SEQ ID NO: 39.

In other aspects of this embodiment, a binding domain comprising a FGF-9β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four non-contiguous amino acid substitutions relative to aminoacids 101-103 or amino acids 145-147 of SEQ ID NO: 39. In other aspectsof this embodiment, a binding domain comprising a FGF-9 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 101-103or amino acids 145-147 of SEQ ID NO: 39. In other aspects of thisembodiment, a non-contiguous amino acid substitution of any amino acidfrom amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39 can bereplaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39 can bereplaced with phenylalanine. In yet other aspects of this embodiment, abinding domain comprising a FGF-9 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 101-103 or amino acids145-147 of SEQ ID NO: 39. In other aspects of this embodiment, a bindingdomain comprising a FGF-9 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino aciddeletions relative to amino acids 101-103 or amino acids 145-147 of SEQID NO: 39. In still other aspects of this embodiment, a binding domaincomprising a FGF-9 β-trefoil domain comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino acidadditions relative to amino acids 101-103 or amino acids 145-147 of SEQID NO: 39. In other aspects of this embodiment, a binding domaincomprising a FGF-9 β-trefoil domain comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 101-103 or amino acids 145-147 of SEQID NO: 39.

In other aspects of this embodiment, a binding domain comprising a FGF-9β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid substitutions relative to aminoacids 101-103 or amino acids 145-147 of SEQ ID NO: 39. In other aspectsof this embodiment, a binding domain comprising a FGF-9 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 101-103 oramino acids 145-147 of SEQ ID NO: 39. In other aspects of thisembodiment, contiguous amino acid substitutions of amino acids fromamino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39 can bereplaced with glycine. In other aspects of this embodiment, contiguousamino acid substitutions of hydrophobic amino acids from amino acids101-103 or amino acids 145-147 of SEQ ID NO: 39 can be replaced withphenylalanine. In yet other aspects of this embodiment, a binding domaincomprising a FGF-9 β-trefoil domain comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 39.In other aspects of this embodiment, a binding domain comprising a FGF-9β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four contiguous amino acid deletions relative to aminoacids 101-103 or amino acids 145-147 of SEQ ID NO: 39. In still otheraspects of this embodiment, a binding domain comprising a FGF-9β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids101-103 or amino acids 145-147 of SEQ ID NO: 39. In other aspects ofthis embodiment, a binding domain comprising a FGF-9 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 101-103 or aminoacids 145-147 of SEQ ID NO: 39.

In another embodiment, a binding domain comprises β-trefoil domainderived a FGF-17 of SEQ ID NO: 40. In another embodiment, a bindingdomain comprising a β-trefoil domain derived from a FGF-17 comprisesamino acids 55-183 of SEQ ID NO: 40. In another aspect of thisembodiment, a binding domain comprising a β-trefoil domain derived froma FGF-17 comprises an α-fold motif of a β-trefoil domain of a FGF-17, aβ-fold motif of a β-trefoil domain of a FGF-17 or a γ-fold motif of aβ-trefoil domain of a FGF-17 of SEQ ID NO: 40. In another aspect of thisembodiment, a β-trefoil domain derived from a FGF-17 comprises aminoacids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO:40.

In other aspects of this embodiment, a binding domain comprising aFGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least70% amino acid identity with amino acids 55-91, amino acids 95-134, oramino acids 138-183 of SEQ ID NO: 40, at least 75% amino acid identitywith amino acids 55-91, amino acids 95-134, or amino acids 138-183 ofSEQ ID NO: 40, at least 80% amino acid identity with amino acids 55-91,amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40, at least85% amino acid identity with amino acids 55-91, amino acids 95-134, oramino acids 138-183 of SEQ ID NO: 40, at least 90% amino acid identitywith amino acids 55-91, amino acids 95-134, or amino acids 138-183 ofSEQ ID NO: 40 or at least 95% amino acid identity with amino acids55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40. Inyet other aspects of this embodiment, a binding domain comprising aFGF-17 β-trefoil domain comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 55-91, amino acids 95-134, oramino acids 138-183 of SEQ ID NO: 40, at most 75% amino acid identitywith amino acids 55-91, amino acids 95-134, or amino acids 138-183 ofSEQ ID NO: 40, at most 80% amino acid identity with amino acids 55-91,amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40, at most 85%amino acid identity with amino acids 55-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 40, at most 90% amino acid identity withamino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ IDNO: 40 or at most 95% amino acid identity with amino acids 55-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 40.

In other aspects of this embodiment, a binding domain comprising aFGF-17 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 55-91,amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40. In otheraspects of this embodiment, a binding domain comprising a FGF-17β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 55-91, amino acids95-134, or amino acids 138-183 of SEQ ID NO: 40. In yet other aspects ofthis embodiment, a binding domain comprising a FGF-17 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 55-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 40. In other aspects of this embodiment, abinding domain comprising a FGF-17 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 55-91, amino acids 95-134, or amino acids138-183 of SEQ ID NO: 40. In still other aspects of this embodiment, abinding domain comprising a FGF-17 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 55-91, amino acids 95-134, or amino acids138-183 of SEQ ID NO: 40. In other aspects of this embodiment, a bindingdomain comprising a FGF-17 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO:40.

In other aspects of this embodiment, a binding domain comprising aFGF-17 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 55-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 40. In other aspectsof this embodiment, a binding domain comprising a FGF-17 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 55-91, amino acids 95-134, oramino acids 138-183 of SEQ ID NO: 40. In yet other aspects of thisembodiment, a binding domain comprising a FGF-17 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 55-91, amino acids 95-134, or amino acids138-183 of SEQ ID NO: 40. In other aspects of this embodiment, a bindingdomain comprising a FGF-17 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 40. Instill other aspects of this embodiment, a binding domain comprising aFGF-17 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 55-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 40. In other aspectsof this embodiment, a binding domain comprising a FGF-17 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 55-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 40.

In another embodiment, a binding domain comprising a FGF-17 β-trefoildomain comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-17or a β8/β9 hairpin turn of a β-trefoil domain of a FGF-17 of SEQ ID NO:35. In another aspect of this embodiment, a binding domain comprising aFGF-17 β-trefoil domain comprises amino acids 92-94 or amino acids135-137 of SEQ ID NO: 40.

In other aspects of this embodiment, a binding domain comprising aFGF-17 β-trefoil domain comprises a polypeptide having, e.g., at least70% amino acid identity with amino acids 92-94 or amino acids 135-137 ofSEQ ID NO: 40, at least 75% amino acid identity with amino acids 92-94or amino acids 135-137 of SEQ ID NO: 40, at least 80% amino acididentity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40,at least 85% amino acid identity with amino acids 92-94 or amino acids135-137 of SEQ ID NO: 40, at least 90% amino acid identity with aminoacids 92-94 or amino acids 135-137 of SEQ ID NO: 40 or at least 95%amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQID NO: 40. In yet other aspects of this embodiment, a binding domaincomprising a FGF-17 β-trefoil domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 92-94 or aminoacids 135-137 of SEQ ID NO: 40, at most 75% amino acid identity withamino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40, at most 80%amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQID NO: 40, at most 85% amino acid identity with amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 40, at most 90% amino acid identitywith amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40 or atmost 95% amino acid identity with amino acids 92-94 or amino acids135-137 of SEQ ID NO: 40.

In other aspects of this embodiment, a binding domain comprising aFGF-17 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three or four non-contiguous amino acid substitutions relativeto amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In otheraspects of this embodiment, a binding domain comprising a FGF-17β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid substitutions relative toamino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany amino acid from amino acids 92-94 or amino acids 135-137 of SEQ IDNO: 40 can be replaced with glycine. In other aspects of thisembodiment, a non-contiguous amino acid substitution of any hydrophobicamino acid from amino acids 92-94 or amino acids 135-137 of SEQ ID NO:40 can be replaced with phenylalanine. In yet other aspects of thisembodiment, a binding domain comprising a FGF-17 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid deletions relative to amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 40. In other aspects of thisembodiment, a binding domain comprising a FGF-17 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid deletions relative to amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 40. In still other aspects of thisembodiment, a binding domain comprising a FGF-17 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid additions relative to amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 40. In other aspects of thisembodiment, a binding domain comprising a FGF-17 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid additions relative to amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 40.

In other aspects of this embodiment, a binding domain comprising aFGF-17 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid substitutions relative toamino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In otheraspects of this embodiment, a binding domain comprising a FGF-17β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four contiguous amino acid substitutions relative to aminoacids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In other aspects ofthis embodiment, contiguous amino acid substitutions of amino acids fromamino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40 can bereplaced with glycine. In other aspects of this embodiment, contiguousamino acid substitutions of hydrophobic amino acids from amino acids92-94 or amino acids 135-137 of SEQ ID NO: 40 can be replaced withphenylalanine. In yet other aspects of this embodiment, a binding domaincomprising a FGF-17 β-trefoil domain comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40.In other aspects of this embodiment, a binding domain comprising aFGF-17 β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three or four contiguous amino acid deletions relative toamino acids 92-94 or amino acids 135-137 of SEQ ID NO: 40. In stillother aspects of this embodiment, a binding domain comprising a FGF-17β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids92-94 or amino acids 135-137 of SEQ ID NO: 40. In other aspects of thisembodiment, a binding domain comprising a FGF-17 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 92-94 or aminoacids 135-137 of SEQ ID NO: 40.

In another embodiment, a binding domain comprises β-trefoil domainderived a FGF-18 of SEQ ID NO: 41. In another embodiment, a bindingdomain comprising a β-trefoil domain derived from a FGF-18 comprisesamino acids 54-183 of SEQ ID NO: 41. In another aspect of thisembodiment, a binding domain comprising a β-trefoil domain derived froma FGF-18 comprises an α-fold motif of a β-trefoil domain of a FGF-18, aβ-fold motif of a β-trefoil domain of a FGF-18 or a γ-fold motif of aβ-trefoil domain of a FGF-18 of SEQ ID NO: 41. In another aspect of thisembodiment, a β-trefoil domain derived from a FGF-18 comprises aminoacids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO:41.

In other aspects of this embodiment, a binding domain comprising aFGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least70% amino acid identity with amino acids 55-91, amino acids 95-134, oramino acids 138-183 of SEQ ID NO: 41, at least 75% amino acid identitywith amino acids 55-91, amino acids 95-134, or amino acids 138-183 ofSEQ ID NO: 41, at least 80% amino acid identity with amino acids 55-91,amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41, at least85% amino acid identity with amino acids 55-91, amino acids 95-134, oramino acids 138-183 of SEQ ID NO: 41, at least 90% amino acid identitywith amino acids 55-91, amino acids 95-134, or amino acids 138-183 ofSEQ ID NO: 41 or at least 95% amino acid identity with amino acids55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41. Inyet other aspects of this embodiment, a binding domain comprising aFGF-18 β-trefoil domain comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 55-91, amino acids 95-134, oramino acids 138-183 of SEQ ID NO: 41, at most 75% amino acid identitywith amino acids 55-91, amino acids 95-134, or amino acids 138-183 ofSEQ ID NO: 41, at most 80% amino acid identity with amino acids 55-91,amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41, at most 85%amino acid identity with amino acids 55-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 41, at most 90% amino acid identity withamino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ IDNO: 41 or at most 95% amino acid identity with amino acids 55-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 41.

In other aspects of this embodiment, a binding domain comprising aFGF-18 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 55-91,amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41. In otheraspects of this embodiment, a binding domain comprising a FGF-18β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 55-91, amino acids95-134, or amino acids 138-183 of SEQ ID NO: 41. In yet other aspects ofthis embodiment, a binding domain comprising a FGF-18 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 55-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 41. In other aspects of this embodiment, abinding domain comprising a FGF-18 β-trefoil domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 55-91, amino acids 95-134, or amino acids138-183 of SEQ ID NO: 41. In still other aspects of this embodiment, abinding domain comprising a FGF-18 β-trefoil domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 55-91, amino acids 95-134, or amino acids138-183 of SEQ ID NO: 41. In other aspects of this embodiment, a bindingdomain comprising a FGF-18 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO:41.

In other aspects of this embodiment, a binding domain comprising aFGF-18 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 55-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 41. In other aspectsof this embodiment, a binding domain comprising a FGF-18 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 55-91, amino acids 95-134, oramino acids 138-183 of SEQ ID NO: 41. In yet other aspects of thisembodiment, a binding domain comprising a FGF-18 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 55-91, amino acids 95-134, or amino acids138-183 of SEQ ID NO: 41. In other aspects of this embodiment, a bindingdomain comprising a FGF-18 β-trefoil domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 41. Instill other aspects of this embodiment, a binding domain comprising aFGF-18 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 55-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 41. In other aspectsof this embodiment, a binding domain comprising a FGF-18 β-trefoildomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 55-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 41.

In another embodiment, a binding domain comprising a FGF-18 β-trefoildomain comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-18or a β8/β9 hairpin turn of a β-trefoil domain of a FGF-18 of SEQ ID NO:35. In another aspect of this embodiment, a binding domain comprising aFGF-18 β-trefoil domain comprises amino acids 92-94 or amino acids135-137 of SEQ ID NO: 41.

In other aspects of this embodiment, a binding domain comprising aFGF-18 β-trefoil domain comprises a polypeptide having, e.g., at least70% amino acid identity with amino acids 92-94 or amino acids 135-137 ofSEQ ID NO: 41, at least 75% amino acid identity with amino acids 92-94or amino acids 135-137 of SEQ ID NO: 41, at least 80% amino acididentity with amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41,at least 85% amino acid identity with amino acids 92-94 or amino acids135-137 of SEQ ID NO: 41, at least 90% amino acid identity with aminoacids 92-94 or amino acids 135-137 of SEQ ID NO: 41 or at least 95%amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQID NO: 41. In yet other aspects of this embodiment, a binding domaincomprising a FGF-18 β-trefoil domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 92-94 or aminoacids 135-137 of SEQ ID NO: 41, at most 75% amino acid identity withamino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41, at most 80%amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQID NO: 41, at most 85% amino acid identity with amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 41, at most 90% amino acid identitywith amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41 or atmost 95% amino acid identity with amino acids 92-94 or amino acids135-137 of SEQ ID NO: 41.

In other aspects of this embodiment, a binding domain comprising aFGF-18 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three or four non-contiguous amino acid substitutions relativeto amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In otheraspects of this embodiment, a binding domain comprising a FGF-18β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid substitutions relative toamino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany amino acid from amino acids 92-94 or amino acids 135-137 of SEQ IDNO: 41 can be replaced with glycine. In other aspects of thisembodiment, a non-contiguous amino acid substitution of any hydrophobicamino acid from amino acids 92-94 or amino acids 135-137 of SEQ ID NO:41 can be replaced with phenylalanine. In yet other aspects of thisembodiment, a binding domain comprising a FGF-18 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid deletions relative to amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 41. In other aspects of thisembodiment, a binding domain comprising a FGF-18 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid deletions relative to amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 41. In still other aspects of thisembodiment, a binding domain comprising a FGF-18 β-trefoil domaincomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid additions relative to amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 41. In other aspects of thisembodiment, a binding domain comprising a FGF-18 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid additions relative to amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 41.

In other aspects of this embodiment, a binding domain comprising aFGF-18 β-trefoil domain comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid substitutions relative toamino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In otheraspects of this embodiment, a binding domain comprising a FGF-18β-trefoil domain comprises a polypeptide having, e.g., at least one,two, three or four contiguous amino acid substitutions relative to aminoacids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In other aspects ofthis embodiment, contiguous amino acid substitutions of amino acids fromamino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41 can bereplaced with glycine. In other aspects of this embodiment, contiguousamino acid substitutions of hydrophobic amino acids from amino acids92-94 or amino acids 135-137 of SEQ ID NO: 41 can be replaced withphenylalanine. In yet other aspects of this embodiment, a binding domaincomprising a FGF-18 β-trefoil domain comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41.In other aspects of this embodiment, a binding domain comprising aFGF-18 β-trefoil domain comprises a polypeptide having, e.g., at leastone, two, three or four contiguous amino acid deletions relative toamino acids 92-94 or amino acids 135-137 of SEQ ID NO: 41. In stillother aspects of this embodiment, a binding domain comprising a FGF-18β-trefoil domain comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids92-94 or amino acids 135-137 of SEQ ID NO: 41. In other aspects of thisembodiment, a binding domain comprising a FGF-18 β-trefoil domaincomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 92-94 or aminoacids 135-137 of SEQ ID NO: 41.

Because of the modular nature of the β-trefoil domain, it is envisionedthat any combination of α-folds, β-folds, γ-folds, β4/β5 β-hairpinturns, β8/β9 β-hairpin turns, or any combination thereof from aβ-trefoil domain of a Clostridial toxin binding domain, a β-trefoildomain of a NAP or a β-trefoil domain, a β-trefoil domain of an FGFligand, or any combination thereof, can be used to practice aspect ofthe present invention. As a non-limiting example, a multivalentClostridial toxin disclosed in the present specification can comprise abinding domain comprising a α-fold from a BoNT/A binding domain, aβ-fold from a FGF-18 and a 2γ-fold from a BoNT/A HA-33. As anothernon-limiting example, a multivalent Clostridial toxin disclosed in thepresent specification can comprise a binding domain comprising a α-foldfrom a BoNT/A HA-17, a β-fold from a Clostridial botulinum serotype ENTNH and a γ-fold from a modified BoNT/C1 binding domain with enhancedbinding activity.

Thus, in an embodiment, a binding domain comprises a glycogen-likepeptide. In another embodiment, a binding domain comprises aglycogen-like peptide of SEQ ID NO: 42. In another embodiment, a bindingdomain is derived from a glycogen-like peptide. In another embodiment, abinding domain is derived from a glycogen-like peptide of SEQ ID NO: 42.In aspects of this embodiment, a binding domain is derived from aglycogen-like peptide comprises a GRPP, a GLP-1, a GLP-2, a glucagon oran oxyntomodulin. In aspects of this embodiment, a binding domain isderived from a glycogen-like peptide comprising amino acids 21-50, aminoacids 53-81, amino acids 53-89, amino acids 98-124, or amino acids146-178 of SEQ ID NO: 42.

In other aspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124,or amino acids 146-178 of SEQ ID NO: 42, at least 75% amino acididentity with amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42, at least80% amino acid identity with amino acids 21-50, amino acids 53-81, aminoacids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO:42, at least 85% amino acid identity with amino acids 21-50, amino acids53-81, amino acids 53-89, amino acids 98-124, or amino acids 146-178 ofSEQ ID NO: 42, at least 90% amino acid identity with amino acids 21-50,amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids146-178 of SEQ ID NO: 42 or at least 95% amino acid identity with aminoacids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124,or amino acids 146-178 of SEQ ID NO: 42. In yet other aspects of thisembodiment, a glycogen-like peptide comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 21-50, aminoacids 53-81, amino acids 53-89, amino acids 98-124, or amino acids146-178 of SEQ ID NO: 42, at most 75% amino acid identity with aminoacids 21-50, amino acids 53-81, amino acids 53-89, amino acids 98-124,or amino acids 146-178 of SEQ ID NO: 42, at most 80% amino acid identitywith amino acids 21-50, amino acids 53-81, amino acids 53-89, aminoacids 98-124, or amino acids 146-178 of SEQ ID NO: 42, at most 85% aminoacid identity with amino acids 21-50, amino acids 53-81, amino acids53-89, amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42, atmost 90% amino acid identity with amino acids 21-50, amino acids 53-81,amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ IDNO: 42 or at most 95% amino acid identity with amino acids 21-50, aminoacids 53-81, amino acids 53-89, amino acids 98-124, or amino acids146-178 of SEQ ID NO: 42.

In other aspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In otheraspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In yetother aspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In otheraspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In stillother aspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In otheraspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42.

In other aspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In otheraspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42. In yetother aspects of this embodiment, a glycogen-like peptide comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 21-50, amino acids 53-81, amino acids 53-89, amino acids98-124, or amino acids 146-178 of SEQ ID NO: 42. In other aspects ofthis embodiment, a glycogen-like peptide comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 21-50,amino acids 53-81, amino acids 53-89, amino acids 98-124, or amino acids146-178 of SEQ ID NO: 42. In still other aspects of this embodiment, aglycogen-like peptide comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 21-50, amino acids 53-81,amino acids 53-89, amino acids 98-124, or amino acids 146-178 of SEQ IDNO: 42. In other aspects of this embodiment, a glycogen-like peptidecomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 21-50, amino acids 53-81, amino acids 53-89,amino acids 98-124, or amino acids 146-178 of SEQ ID NO: 42.

In an embodiment, a binding domain comprises a PACAP. In anotherembodiment, a binding domain comprises a PACAP of SEQ ID NO: 43. Inanother embodiment, a binding domain is derived from a PACAP. In anotherembodiment, a binding domain is derived from a PACAP of SEQ ID NO: 43.In an aspect of this embodiment, a binding domain is derived from aPACAP comprising amino acids 132-158 of SEQ ID NO: 43.

In other aspects of this embodiment, a PACAP comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 132-158of SEQ ID NO: 43, at least 75% amino acid identity with amino acids132-158 of SEQ ID NO: 43, at least 80% amino acid identity with aminoacids 132-158 of SEQ ID NO: 43, at least 85% amino acid identity withamino acids 132-158 of SEQ ID NO: 43, at least 90% amino acid identitywith amino acids 132-158 of SEQ ID NO: 43 or at least 95% amino acididentity with amino acids 132-158 of SEQ ID NO: 43. In yet other aspectsof this embodiment, a PACAP comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 132-158 of SEQ ID NO: 43,at most 75% amino acid identity with amino acids 132-158 of SEQ ID NO:43, at most 80% amino acid identity with amino acids 132-158 of SEQ IDNO: 43, at most 85% amino acid identity with amino acids 132-158 of SEQID NO: 43, at most 90% amino acid identity with amino acids 132-158 ofSEQ ID NO: 43 or at most 95% amino acid identity with amino acids132-158 of SEQ ID NO: 43.

In other aspects of this embodiment, a PACAP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 132-158 of SEQ ID NO: 43. In other aspects of this embodiment, aPACAP comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 132-158 of SEQ ID NO: 43. In yetother aspects of this embodiment, a PACAP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 132-158 of SEQ ID NO: 43. In other aspects of this embodiment, aPACAP comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 132-158 of SEQ ID NO: 43. In stillother aspects of this embodiment, a PACAP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 132-158 of SEQ ID NO: 43. In other aspects of this embodiment, aPACAP comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 132-158 of SEQ ID NO: 43.

In other aspects of this embodiment, a PACAP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 132-158 of SEQ ID NO: 43. In other aspects of this embodiment, aPACAP comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 132-158 of SEQ ID NO: 43. In yetother aspects of this embodiment, a PACAP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids132-158 of SEQ ID NO: 43. In other aspects of this embodiment, a PACAPcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 132-158 of SEQ ID NO: 43. In still other aspectsof this embodiment, a PACAP comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 132-158 of SEQID NO: 43. In other aspects of this embodiment, a PACAP comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 132-158 of SEQ ID NO: 43.

In an embodiment, a binding domain comprises a GHRH. In anotherembodiment, a binding domain comprises a GHRH of SEQ ID NO: 44. Inanother embodiment, a binding domain is derived from a GHRH. In anotherembodiment, a binding domain is derived from a GHRH of SEQ ID NO: 44. Inaspects of this embodiment, a binding domain is derived from a GHRHcomprising amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44.

In other aspects of this embodiment, a GHRH comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 32-58 oramino acids 32-75 of SEQ ID NO: 44, at least 75% amino acid identitywith amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44, at least80% amino acid identity with amino acids 32-58 or amino acids 32-75 ofSEQ ID NO: 44, at least 85% amino acid identity with amino acids 32-58or amino acids 32-75 of SEQ ID NO: 44, at least 90% amino acid identitywith amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44 or at least95% amino acid identity with amino acids 32-58 or amino acids 32-75 ofSEQ ID NO: 44. In yet other aspects of this embodiment, a GHRH comprisesa polypeptide having, e.g., at most 70% amino acid identity with aminoacids 32-58 or amino acids 32-75 of SEQ ID NO: 44, at most 75% aminoacid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO:44, at most 80% amino acid identity with amino acids 32-58 or aminoacids 32-75 of SEQ ID NO: 44, at most 85% amino acid identity with aminoacids 32-58 or amino acids 32-75 of SEQ ID NO: 44, at most 90% aminoacid identity with amino acids 32-58 or amino acids 32-75 of SEQ ID NO:44 or at most 95% amino acid identity with amino acids 32-58 or aminoacids 32-75 of SEQ ID NO: 44.

In other aspects of this embodiment, a GHRH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 32-58 or amino acids 32-75 of SEQ ID NO: 44. In other aspects ofthis embodiment, a GHRH comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 32-58 oramino acids 32-75 of SEQ ID NO: 44. In yet other aspects of thisembodiment, a GHRH comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 32-58 or amino acids 32-75of SEQ ID NO: 44. In other aspects of this embodiment, a GHRH comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44. Instill other aspects of this embodiment, a GHRH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 32-58 or amino acids 32-75 of SEQ ID NO: 44. In other aspects ofthis embodiment, a GHRH comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 32-58 oramino acids 32-75 of SEQ ID NO: 44.

In other aspects of this embodiment, a GHRH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 32-58 or amino acids 32-75 of SEQ ID NO: 44. In other aspects ofthis embodiment, a GHRH comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 32-58 oramino acids 32-75 of SEQ ID NO: 44. In yet other aspects of thisembodiment, a GHRH comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 32-58 or amino acids 32-75of SEQ ID NO: 44. In other aspects of this embodiment, a GHRH comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 32-58 or amino acids 32-75 of SEQ ID NO: 44. In still otheraspects of this embodiment, a GHRH comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 32-58 or aminoacids 32-75 of SEQ ID NO: 44. In other aspects of this embodiment, aGHRH comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 32-58 or amino acids 32-75 of SEQ IDNO: 44.

In an embodiment, a binding domain comprises a VIP1. In anotherembodiment, a binding domain comprises a VIP1 of SEQ ID NO: 45. Inanother embodiment, a binding domain is derived from a VIP1. In anotherembodiment, a binding domain is derived from a VIP1 of SEQ ID NO: 45. Inaspects of this embodiment, a binding domain is derived from a VIP1comprising amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45.

In other aspects of this embodiment, a VIP1 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 81-107or amino acids 125-151 of SEQ ID NO: 45, at least 75% amino acididentity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO:45, at least 80% amino acid identity with amino acids 81-107 or aminoacids 125-151 of SEQ ID NO: 45, at least 85% amino acid identity withamino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45, at least 90%amino acid identity with amino acids 81-107 or amino acids 125-151 ofSEQ ID NO: 45 or at least 95% amino acid identity with amino acids81-107 or amino acids 125-151 of SEQ ID NO: 45. In yet other aspects ofthis embodiment, a VIP1 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 81-107 or amino acids 125-151of SEQ ID NO: 45, at most 75% amino acid identity with amino acids81-107 or amino acids 125-151 of SEQ ID NO: 45, at most 80% amino acididentity with amino acids 81-107 or amino acids 125-151 of SEQ ID NO:45, at most 85% amino acid identity with amino acids 81-107 or aminoacids 125-151 of SEQ ID NO: 45, at most 90% amino acid identity withamino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45 or at most95% amino acid identity with amino acids 81-107 or amino acids 125-151of SEQ ID NO: 45.

In other aspects of this embodiment, a VIP1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 81-107 or amino acids 125-151 of SEQ ID NO: 45. In other aspectsof this embodiment, a VIP1 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 81-107or amino acids 125-151 of SEQ ID NO: 45. In yet other aspects of thisembodiment, a VIP1 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 81-107 or amino acids125-151 of SEQ ID NO: 45. In other aspects of this embodiment, a VIP1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 81-107 or amino acids 125-151 of SEQID NO: 45. In still other aspects of this embodiment, a VIP1 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45.In other aspects of this embodiment, a VIP1 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 81-107 or amino acids 125-151 of SEQ ID NO: 45.

In other aspects of this embodiment, a VIP1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 81-107 or amino acids 125-151 of SEQ ID NO: 45. In other aspectsof this embodiment, a VIP1 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 81-107 oramino acids 125-151 of SEQ ID NO: 45. In yet other aspects of thisembodiment, a VIP1 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 81-107 or amino acids125-151 of SEQ ID NO: 45. In other aspects of this embodiment, a VIP1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45.In still other aspects of this embodiment, a VIP1 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 81-107 or amino acids 125-151 of SEQ ID NO: 45. In otheraspects of this embodiment, a VIP1 comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 81-107 or aminoacids 125-151 of SEQ ID NO: 45.

In an embodiment, a binding domain comprises a VIP2. In anotherembodiment, a binding domain comprises a VIP2 of SEQ ID NO: 46. Inanother embodiment, a binding domain is derived from a VIP2. In anotherembodiment, a binding domain is derived from a VIP2 of SEQ ID NO: 46. Inaspects of this embodiment, a binding domain is derived from a VIP2comprising amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46.

In other aspects of this embodiment, a VIP2 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 81-107or amino acids 124-150 of SEQ ID NO: 46, at least 75% amino acididentity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO:46, at least 80% amino acid identity with amino acids 81-107 or aminoacids 124-150 of SEQ ID NO: 46, at least 85% amino acid identity withamino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46, at least 90%amino acid identity with amino acids 81-107 or amino acids 124-150 ofSEQ ID NO: 46 or at least 95% amino acid identity with amino acids81-107 or amino acids 124-150 of SEQ ID NO: 46. In yet other aspects ofthis embodiment, a VIP2 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 81-107 or amino acids 124-150of SEQ ID NO: 46, at most 75% amino acid identity with amino acids81-107 or amino acids 124-150 of SEQ ID NO: 46, at most 80% amino acididentity with amino acids 81-107 or amino acids 124-150 of SEQ ID NO:46, at most 85% amino acid identity with amino acids 81-107 or aminoacids 124-150 of SEQ ID NO: 46, at most 90% amino acid identity withamino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46 or at most95% amino acid identity with amino acids 81-107 or amino acids 124-150of SEQ ID NO: 46.

In other aspects of this embodiment, a VIP2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 81-107 or amino acids 124-150 of SEQ ID NO: 46. In other aspectsof this embodiment, a VIP2 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 81-107or amino acids 124-150 of SEQ ID NO: 46. In yet other aspects of thisembodiment, a VIP2 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 81-107 or amino acids124-150 of SEQ ID NO: 46. In other aspects of this embodiment, a VIP2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 81-107 or amino acids 124-150 of SEQID NO: 46. In still other aspects of this embodiment, a VIP2 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46.In other aspects of this embodiment, a VIP2 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 81-107 or amino acids 124-150 of SEQ ID NO: 46.

In other aspects of this embodiment, a VIP2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 81-107 or amino acids 124-150 of SEQ ID NO: 46. In other aspectsof this embodiment, a VIP2 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 81-107 oramino acids 124-150 of SEQ ID NO: 46. In yet other aspects of thisembodiment, a VIP2 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 81-107 or amino acids124-150 of SEQ ID NO: 46. In other aspects of this embodiment, a VIP2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46.In still other aspects of this embodiment, a VIP2 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 81-107 or amino acids 124-150 of SEQ ID NO: 46. In otheraspects of this embodiment, a VIP2 comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 81-107 or aminoacids 124-150 of SEQ ID NO: 46.

In an embodiment, a binding domain comprises a GIP. In anotherembodiment, a binding domain comprises a GIP of SEQ ID NO: 47. Inanother embodiment, a binding domain is derived from a GIP. In anotherembodiment, a binding domain is derived from a GIP of SEQ ID NO: 47. Inaspects of this embodiment, a binding domain is derived from a GIPcomprising amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47.

In other aspects of this embodiment, a GIP comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 52-78 oramino acids 52-93 of SEQ ID NO: 47, at least 75% amino acid identitywith amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47, at least80% amino acid identity with amino acids 52-78 or amino acids 52-93 ofSEQ ID NO: 47, at least 85% amino acid identity with amino acids 52-78or amino acids 52-93 of SEQ ID NO: 47, at least 90% amino acid identitywith amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47 or at least95% amino acid identity with amino acids 52-78 or amino acids 52-93 ofSEQ ID NO: 47. In yet other aspects of this embodiment, a GIP comprisesa polypeptide having, e.g., at most 70% amino acid identity with aminoacids 52-78 or amino acids 52-93 of SEQ ID NO: 47, at most 75% aminoacid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO:47, at most 80% amino acid identity with amino acids 52-78 or aminoacids 52-93 of SEQ ID NO: 47, at most 85% amino acid identity with aminoacids 52-78 or amino acids 52-93 of SEQ ID NO: 47, at most 90% aminoacid identity with amino acids 52-78 or amino acids 52-93 of SEQ ID NO:47 or at most 95% amino acid identity with amino acids 52-78 or aminoacids 52-93 of SEQ ID NO: 47.

In other aspects of this embodiment, a GIP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 52-78 or amino acids 52-93 of SEQ ID NO: 47. In other aspects ofthis embodiment, a GIP comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 52-78 oramino acids 52-93 of SEQ ID NO: 47. In yet other aspects of thisembodiment, a GIP comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 52-78 or amino acids 52-93of SEQ ID NO: 47. In other aspects of this embodiment, a GIP comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47. Instill other aspects of this embodiment, a GIP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 52-78 or amino acids 52-93 of SEQ ID NO: 47. In other aspects ofthis embodiment, a GIP comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 52-78 oramino acids 52-93 of SEQ ID NO: 47.

In other aspects of this embodiment, a GIP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 52-78 or amino acids 52-93 of SEQ ID NO: 47. In other aspects ofthis embodiment, a GIP comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 52-78 oramino acids 52-93 of SEQ ID NO: 47. In yet other aspects of thisembodiment, a GIP comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 52-78 or amino acids 52-93of SEQ ID NO: 47. In other aspects of this embodiment, a GIP comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47. In still otheraspects of this embodiment, a GIP comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 52-78 or aminoacids 52-93 of SEQ ID NO: 47. In other aspects of this embodiment, a GIPcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 52-78 or amino acids 52-93 of SEQ ID NO: 47.

In an embodiment, a binding domain comprises a Secretin. In anotherembodiment, a binding domain comprises a Secretin of SEQ ID NO: 48. Inanother embodiment, a binding domain is derived from a Secretin. Inanother embodiment, a binding domain is derived from a Secretin of SEQID NO: 48. In an aspect of this embodiment, a binding domain is derivedfrom a Secretin comprising amino acids 28-54 of SEQ ID NO: 48.

In other aspects of this embodiment, a Secretin comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 28-54 ofSEQ ID NO: 48, at least 75% amino acid identity with amino acids 28-54of SEQ ID NO: 48, at least 80% amino acid identity with amino acids28-54 of SEQ ID NO: 48, at least 85% amino acid identity with aminoacids 28-54 of SEQ ID NO: 48, at least 90% amino acid identity withamino acids 28-54 of SEQ ID NO: 48 or at least 95% amino acid identitywith amino acids 28-54 of SEQ ID NO: 48. In yet other aspects of thisembodiment, a Secretin comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 28-54 of SEQ ID NO: 48, at most 75%amino acid identity with amino acids 28-54 of SEQ ID NO: 48, at most 80%amino acid identity with amino acids 28-54 of SEQ ID NO: 48, at most 85%amino acid identity with amino acids 28-54 of SEQ ID NO: 48, at most 90%amino acid identity with amino acids 28-54 of SEQ ID NO: 48 or at most95% amino acid identity with amino acids 28-54 of SEQ ID NO: 48.

In other aspects of this embodiment, a Secretin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 28-54 of SEQ ID NO: 48. In other aspects of this embodiment, aSecretin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 28-54 of SEQ ID NO: 48. In yetother aspects of this embodiment, a Secretin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 28-54 of SEQ ID NO: 48. In other aspects of this embodiment, aSecretin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 28-54 of SEQ ID NO: 48. In still otheraspects of this embodiment, a Secretin comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids 28-54of SEQ ID NO: 48. In other aspects of this embodiment, a Secretincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 28-54 of SEQ ID NO: 48.

In other aspects of this embodiment, a Secretin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 28-54 of SEQ ID NO: 48. In other aspects of this embodiment, aSecretin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 28-54 of SEQ ID NO: 48. In yetother aspects of this embodiment, a Secretin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids28-54 of SEQ ID NO: 48. In other aspects of this embodiment, a Secretincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 28-54 of SEQ ID NO: 48. In still other aspectsof this embodiment, a Secretin comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 28-54 of SEQ IDNO: 48. In other aspects of this embodiment, a Secretin comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 28-54 of SEQ ID NO: 48.

In an embodiment, a binding domain comprises a Gastrin. In anotherembodiment, a binding domain comprises a Gastrin of SEQ ID NO: 49. Inanother embodiment, a binding domain is derived from a Gastrin. Inanother embodiment, a binding domain is derived from a Gastrin of SEQ IDNO: 49. In aspects of this embodiment, a binding domain is derived froma Gastrin comprising amino acids 76-92 or amino acids 59-92 of SEQ IDNO: 49.

In other aspects of this embodiment, a Gastrin comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 76-92 oramino acids 59-92 of SEQ ID NO: 49, at least 75% amino acid identitywith amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49, at least80% amino acid identity with amino acids 76-92 or amino acids 59-92 ofSEQ ID NO: 49, at least 85% amino acid identity with amino acids 76-92or amino acids 59-92 of SEQ ID NO: 49, at least 90% amino acid identitywith amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49 or at least95% amino acid identity with amino acids 76-92 or amino acids 59-92 ofSEQ ID NO: 49. In yet other aspects of this embodiment, a Gastrincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49, at most75% amino acid identity with amino acids 76-92 or amino acids 59-92 ofSEQ ID NO: 49, at most 80% amino acid identity with amino acids 76-92 oramino acids 59-92 of SEQ ID NO: 49, at most 85% amino acid identity withamino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49, at most 90%amino acid identity with amino acids 76-92 or amino acids 59-92 of SEQID NO: 49 or at most 95% amino acid identity with amino acids 76-92 oramino acids 59-92 of SEQ ID NO: 49.

In other aspects of this embodiment, a Gastrin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 76-92 or amino acids 59-92 of SEQ ID NO: 49. In other aspects ofthis embodiment, a Gastrin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 76-92 oramino acids 59-92 of SEQ ID NO: 49. In yet other aspects of thisembodiment, a Gastrin comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 76-92 or amino acids 59-92of SEQ ID NO: 49. In other aspects of this embodiment, a Gastrincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 76-92 or amino acids 59-92 of SEQ IDNO: 49. In still other aspects of this embodiment, a Gastrin comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49. Inother aspects of this embodiment, a Gastrin comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 76-92 or amino acids 59-92 of SEQ ID NO: 49.

In other aspects of this embodiment, a Gastrin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 76-92 or amino acids 59-92 of SEQ ID NO: 49. In other aspects ofthis embodiment, a Gastrin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 76-92 oramino acids 59-92 of SEQ ID NO: 49. In yet other aspects of thisembodiment, a Gastrin comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 76-92 or amino acids 59-92of SEQ ID NO: 49. In other aspects of this embodiment, a Gastrincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49. Instill other aspects of this embodiment, a Gastrin comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 76-92 or amino acids 59-92 of SEQ ID NO: 49. In otheraspects of this embodiment, a Gastrin comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 76-92 oramino acids 59-92 of SEQ ID NO: 49.

In an embodiment, a binding domain comprises a GRP. In anotherembodiment, a binding domain comprises a GRP of SEQ ID NO: 50. Inanother embodiment, a binding domain is derived from a GRP. In anotherembodiment, a binding domain is derived from a GRP of SEQ ID NO: 50. Inaspects of this embodiment, a binding domain is derived from a GRPcomprising amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50.

In other aspects of this embodiment, a GRP comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 41-50 oramino acids 24-50 of SEQ ID NO: 50, at least 75% amino acid identitywith amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50, at least80% amino acid identity with amino acids 41-50 or amino acids 24-50 ofSEQ ID NO: 50, at least 85% amino acid identity with amino acids 41-50or amino acids 24-50 of SEQ ID NO: 50, at least 90% amino acid identitywith amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50 or at least95% amino acid identity with amino acids 41-50 or amino acids 24-50 ofSEQ ID NO: 50. In yet other aspects of this embodiment, a GRP comprisesa polypeptide having, e.g., at most 70% amino acid identity with aminoacids 41-50 or amino acids 24-50 of SEQ ID NO: 50, at most 75% aminoacid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO:50, at most 80% amino acid identity with amino acids 41-50 or aminoacids 24-50 of SEQ ID NO: 50, at most 85% amino acid identity with aminoacids 41-50 or amino acids 24-50 of SEQ ID NO: 50, at most 90% aminoacid identity with amino acids 41-50 or amino acids 24-50 of SEQ ID NO:50 or at most 95% amino acid identity with amino acids 41-50 or aminoacids 24-50 of SEQ ID NO: 50.

In other aspects of this embodiment, a GRP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 41-50 or amino acids 24-50 of SEQ ID NO: 50. In other aspects ofthis embodiment, a GRP comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 41-50 oramino acids 24-50 of SEQ ID NO: 50. In yet other aspects of thisembodiment, a GRP comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 41-50 or amino acids 24-50of SEQ ID NO: 50. In other aspects of this embodiment, a GRP comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50. Instill other aspects of this embodiment, a GRP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 41-50 or amino acids 24-50 of SEQ ID NO: 50. In other aspects ofthis embodiment, a GRP comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 41-50 oramino acids 24-50 of SEQ ID NO: 50.

In other aspects of this embodiment, a GRP comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 41-50 or amino acids 24-50 of SEQ ID NO: 50. In other aspects ofthis embodiment, a GRP comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 41-50 oramino acids 24-50 of SEQ ID NO: 50. In yet other aspects of thisembodiment, a GRP comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 41-50 or amino acids 24-50of SEQ ID NO: 50. In other aspects of this embodiment, a GRP comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50. In still otheraspects of this embodiment, a GRP comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 41-50 or aminoacids 24-50 of SEQ ID NO: 50. In other aspects of this embodiment, a GRPcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 41-50 or amino acids 24-50 of SEQ ID NO: 50.

In an embodiment, a binding domain comprises a CCK. In anotherembodiment, a binding domain comprises a CCK of SEQ ID NO: 51. Inanother embodiment, a binding domain is derived from a CCK. In anotherembodiment, a binding domain is derived from a CCK of SEQ ID NO: 51. Inan aspect of this embodiment, a binding domain is derived from a CCKcomprising amino acids 99-112 of SEQ ID NO: 51.

In other aspects of this embodiment, a CCK comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 99-112of SEQ ID NO: 51, at least 75% amino acid identity with amino acids99-112 of SEQ ID NO: 51, at least 80% amino acid identity with aminoacids 99-112 of SEQ ID NO: 51, at least 85% amino acid identity withamino acids 99-112 of SEQ ID NO: 51, at least 90% amino acid identitywith amino acids 99-112 of SEQ ID NO: 51 or at least 95% amino acididentity with amino acids 99-112 of SEQ ID NO: 51. In yet other aspectsof this embodiment, a CCK comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 99-112 of SEQ ID NO: 51, atmost 75% amino acid identity with amino acids 99-112 of SEQ ID NO: 51,at most 80% amino acid identity with amino acids 99-112 of SEQ ID NO:51, at most 85% amino acid identity with amino acids 99-112 of SEQ IDNO: 51, at most 90% amino acid identity with amino acids 99-112 of SEQID NO: 51 or at most 95% amino acid identity with amino acids 99-112 ofSEQ ID NO: 51.

In other aspects of this embodiment, a CCK comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 99-112 of SEQ ID NO: 51. In other aspects of this embodiment, aCCK comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 99-112 of SEQ ID NO: 51. In yetother aspects of this embodiment, a CCK comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids 99-112of SEQ ID NO: 51. In other aspects of this embodiment, a CCK comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 99-112 of SEQ ID NO: 51. In still other aspectsof this embodiment, a CCK comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 99-112 ofSEQ ID NO: 51. In other aspects of this embodiment, a CCK comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 99-112 of SEQ ID NO: 51.

In other aspects of this embodiment, a CCK comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 99-112 of SEQ ID NO: 51. In other aspects of this embodiment, aCCK comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 99-112 of SEQ ID NO: 51. In yetother aspects of this embodiment, a CCK comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 99-112 ofSEQ ID NO: 51. In other aspects of this embodiment, a CCK comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 99-112 of SEQ ID NO: 51. In still other aspects of thisembodiment, a CCK comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 99-112 of SEQ ID NO: 51. Inother aspects of this embodiment, a CCK comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 99-112 ofSEQ ID NO: 51.

Another example of a binding domain disclosed in the presentspecification is, e.g., neurohormones, such as, e.g.,corticotropin-releasing hormone (CCRH) or parathyroid hormone (PTH).

Thus, in an embodiment, a binding domain comprises a CCRH. In anotherembodiment, a binding domain comprises a CCRH of SEQ ID NO: 52. Inanother embodiment, a binding domain is derived from a CCRH. In anotherembodiment, a binding domain is derived from a CCRH of SEQ ID NO: 52. Inaspects of this embodiment, a binding domain is derived from a CCRHcomprising amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52.

In other aspects of this embodiment, a CCRH comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 159-193or amino acids 154-194 of SEQ ID NO: 52, at least 75% amino acididentity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO:52, at least 80% amino acid identity with amino acids 159-193 or aminoacids 154-194 of SEQ ID NO: 52, at least 85% amino acid identity withamino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52, at least90% amino acid identity with amino acids 159-193 or amino acids 154-194of SEQ ID NO: 52 or at least 95% amino acid identity with amino acids159-193 or amino acids 154-194 of SEQ ID NO: 52. In yet other aspects ofthis embodiment, a CCRH comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 159-193 or amino acids 154-194of SEQ ID NO: 52, at most 75% amino acid identity with amino acids159-193 or amino acids 154-194 of SEQ ID NO: 52, at most 80% amino acididentity with amino acids 159-193 or amino acids 154-194 of SEQ ID NO:52, at most 85% amino acid identity with amino acids 159-193 or aminoacids 154-194 of SEQ ID NO: 52, at most 90% amino acid identity withamino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52 or at most95% amino acid identity with amino acids 159-193 or amino acids 154-194of SEQ ID NO: 52.

In other aspects of this embodiment, a CCRH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 159-193 or amino acids 154-194 of SEQ ID NO: 52. In other aspectsof this embodiment, a CCRH comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 159-193or amino acids 154-194 of SEQ ID NO: 52. In yet other aspects of thisembodiment, a CCRH comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 159-193 or amino acids154-194 of SEQ ID NO: 52. In other aspects of this embodiment, a CCRHcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 159-193 or amino acids 154-194 of SEQID NO: 52. In still other aspects of this embodiment, a CCRH comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52.In other aspects of this embodiment, a CCRH comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 159-193 or amino acids 154-194 of SEQ ID NO: 52.

In other aspects of this embodiment, a CCRH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 159-193 or amino acids 154-194 of SEQ ID NO: 52. In other aspectsof this embodiment, a CCRH comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 159-193 oramino acids 154-194 of SEQ ID NO: 52. In yet other aspects of thisembodiment, a CCRH comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 159-193 or amino acids154-194 of SEQ ID NO: 52. In other aspects of this embodiment, a CCRHcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52.In still other aspects of this embodiment, a CCRH comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 159-193 or amino acids 154-194 of SEQ ID NO: 52. In otheraspects of this embodiment, a CCRH comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 159-193 or aminoacids 154-194 of SEQ ID NO: 52.

In an embodiment, a binding domain comprises a PTH. In anotherembodiment, a binding domain comprises a PTH of SEQ ID NO: 53. Inanother embodiment, a binding domain is derived from a PTH. In anotherembodiment, a binding domain is derived from a PTH of SEQ ID NO: 53. Inaspects of this embodiment, a binding domain is derived from a PTHcomprising amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53.

In other aspects of this embodiment, a PTH comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 35-70 oramino acids 145-177 of SEQ ID NO: 53, at least 75% amino acid identitywith amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53, at least80% amino acid identity with amino acids 35-70 or amino acids 145-177 ofSEQ ID NO: 53, at least 85% amino acid identity with amino acids 35-70or amino acids 145-177 of SEQ ID NO: 53, at least 90% amino acididentity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53or at least 95% amino acid identity with amino acids 35-70 or aminoacids 145-177 of SEQ ID NO: 53. In yet other aspects of this embodiment,a PTH comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53,at most 75% amino acid identity with amino acids 35-70 or amino acids145-177 of SEQ ID NO: 53, at most 80% amino acid identity with aminoacids 35-70 or amino acids 145-177 of SEQ ID NO: 53, at most 85% aminoacid identity with amino acids 35-70 or amino acids 145-177 of SEQ IDNO: 53, at most 90% amino acid identity with amino acids 35-70 or aminoacids 145-177 of SEQ ID NO: 53 or at most 95% amino acid identity withamino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53.

In other aspects of this embodiment, a PTH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 35-70 or amino acids 145-177 of SEQ ID NO: 53. In other aspects ofthis embodiment, a PTH comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 35-70 oramino acids 145-177 of SEQ ID NO: 53. In yet other aspects of thisembodiment, a PTH comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 35-70 or amino acids145-177 of SEQ ID NO: 53. In other aspects of this embodiment, a PTHcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 35-70 or amino acids 145-177 of SEQ IDNO: 53. In still other aspects of this embodiment, a PTH comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53.In other aspects of this embodiment, a PTH comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 35-70 or amino acids 145-177 of SEQ ID NO: 53.

In other aspects of this embodiment, a PTH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 35-70 or amino acids 145-177 of SEQ ID NO: 53. In other aspects ofthis embodiment, a PTH comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 35-70 oramino acids 145-177 of SEQ ID NO: 53. In yet other aspects of thisembodiment, a PTH comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 35-70 or amino acids145-177 of SEQ ID NO: 53. In other aspects of this embodiment, a PTHcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 35-70 or amino acids 145-177 of SEQ ID NO: 53.In still other aspects of this embodiment, a PTH comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids35-70 or amino acids 145-177 of SEQ ID NO: 53. In other aspects of thisembodiment, a PTH comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 35-70 or amino acids145-177 of SEQ ID NO: 53.

Another example of a binding domain disclosed in the presentspecification is, e.g., neuroregulatory cytokines, such as, e.g.,ciliary neurotrophic factor (CNTF), glycophorin-A (GPA), leukemiainhibitory factor (LIF), also known as cholinergic differentiationfactor (CDF), interleukins (ILs), like IL1, IL2, IL6, IL8 and IL10,onostatin M, cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC),or neuroleukin, also known as glucose phosphate isomerase (GPI),autocrine motility factor (AMF), maturation and differentiation factor(MF).

Thus, in an embodiment, a binding domain comprises a CNTF. In anotherembodiment, a binding domain comprises a CNTF of SEQ ID NO: 54. Inanother embodiment, a binding domain is derived from a CNTF. In anotherembodiment, a binding domain is derived from a CNTF of SEQ ID NO: 54. Inaspects of this embodiment, a CNTF comprises a polypeptide having, e.g.,at least 70% amino acid identity with the amino acid sequence of SEQ IDNO: 54, at least 75% amino acid identity with the amino acid sequence ofSEQ ID NO: 54, at least 80% amino acid identity with the amino acidsequence of SEQ ID NO: 54, at least 85% amino acid identity with theamino acid sequence of SEQ ID NO: 54, at least 90% amino acid identitywith the amino acid sequence of SEQ ID NO: 54 or at least 95% amino acididentity with the amino acid sequence of SEQ ID NO: 54. In yet otheraspects of this embodiment, a CNTF comprises a polypeptide having, e.g.,at most 70% amino acid identity with the amino acid sequence of SEQ IDNO: 54, at most 75% amino acid identity with the amino acid sequence ofSEQ ID NO: 54, at most 80% amino acid identity with the amino acidsequence of SEQ ID NO: 54, at most 85% amino acid identity with theamino acid sequence of SEQ ID NO: 54, at most 90% amino acid identitywith the amino acid sequence of SEQ ID NO: 54 or at most 95% amino acididentity with the amino acid sequence of SEQ ID NO: 54.

In other aspects of this embodiment, a CNTF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to theamino acid sequence of SEQ ID NO: 54. In other aspects of thisembodiment, a CNTF comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to the amino acid sequence of SEQ IDNO: 54. In yet other aspects of this embodiment, a CNTF comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 54. In other aspectsof this embodiment, a CNTF comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to the amino acid sequenceof SEQ ID NO: 54. In still other aspects of this embodiment, a CNTFcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to the amino acid sequence of SEQ ID NO: 54. In otheraspects of this embodiment, a CNTF comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to the amino acid sequenceof SEQ ID NO: 54.

In other aspects of this embodiment, a CNTF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to the aminoacid sequence of SEQ ID NO: 54. In other aspects of this embodiment, aCNTF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to the amino acid sequence of SEQ ID NO: 54. Inyet other aspects of this embodiment, a CNTF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to the aminoacid sequence of SEQ ID NO: 54. In other aspects of this embodiment, aCNTF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to the amino acid sequence of SEQ ID NO: 54. In stillother aspects of this embodiment, a CNTF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to the amino acidsequence of SEQ ID NO: 54. In other aspects of this embodiment, a CNTFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 54.

In an embodiment, a binding domain comprises a GPA. In anotherembodiment, a binding domain comprises a GPA of SEQ ID NO: 55. Inanother embodiment, a binding domain is derived from a GPA. In anotherembodiment, a binding domain is derived from a GPA of SEQ ID NO: 55. Inaspects of this embodiment, a GPA comprises a polypeptide having, e.g.,at least 70% amino acid identity with the amino acid sequence of SEQ IDNO: 55, at least 75% amino acid identity with the amino acid sequence ofSEQ ID NO: 55, at least 80% amino acid identity with the amino acidsequence of SEQ ID NO: 55, at least 85% amino acid identity with theamino acid sequence of SEQ ID NO: 55, at least 90% amino acid identitywith the amino acid sequence of SEQ ID NO: 55 or at least 95% amino acididentity with the amino acid sequence of SEQ ID NO: 55. In yet otheraspects of this embodiment, a GPA comprises a polypeptide having, e.g.,at most 70% amino acid identity with the amino acid sequence of SEQ IDNO: 55, at most 75% amino acid identity with the amino acid sequence ofSEQ ID NO: 55, at most 80% amino acid identity with the amino acidsequence of SEQ ID NO: 55, at most 85% amino acid identity with theamino acid sequence of SEQ ID NO: 55, at most 90% amino acid identitywith the amino acid sequence of SEQ ID NO: 55 or at most 95% amino acididentity with the amino acid sequence of SEQ ID NO: 55.

In other aspects of this embodiment, a GPA comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to theamino acid sequence of SEQ ID NO: 55. In other aspects of thisembodiment, a GPA comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to the amino acid sequence of SEQ IDNO: 55. In yet other aspects of this embodiment, a GPA comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 55. In other aspectsof this embodiment, a GPA comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to the amino acid sequenceof SEQ ID NO: 55. In still other aspects of this embodiment, a GPAcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to the amino acid sequence of SEQ ID NO: 55. In otheraspects of this embodiment, a GPA comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to the amino acid sequenceof SEQ ID NO: 55.

In other aspects of this embodiment, a GPA comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to the aminoacid sequence of SEQ ID NO: 55. In other aspects of this embodiment, aGPA comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to the amino acid sequence of SEQ ID NO: 55. Inyet other aspects of this embodiment, a GPA comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to the aminoacid sequence of SEQ ID NO: 55. In other aspects of this embodiment, aGPA comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to the amino acid sequence of SEQ ID NO: 55. In stillother aspects of this embodiment, a GPA comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to the amino acidsequence of SEQ ID NO: 55. In other aspects of this embodiment, a GPAcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 55.

In an embodiment, a binding domain comprises a LIF. In anotherembodiment, a binding domain comprises a LIF of SEQ ID NO: 56. Inanother embodiment, a binding domain is derived from a LIF. In anotherembodiment, a binding domain is derived from a LIF of SEQ ID NO: 56. Inaspects of this embodiment, a LIF comprises a polypeptide having, e.g.,at least 70% amino acid identity with the amino acid sequence of SEQ IDNO: 56, at least 75% amino acid identity with the amino acid sequence ofSEQ ID NO: 56, at least 80% amino acid identity with the amino acidsequence of SEQ ID NO: 56, at least 85% amino acid identity with theamino acid sequence of SEQ ID NO: 56, at least 90% amino acid identitywith the amino acid sequence of SEQ ID NO: 56 or at least 95% amino acididentity with the amino acid sequence of SEQ ID NO: 56. In yet otheraspects of this embodiment, a LIF comprises a polypeptide having, e.g.,at most 70% amino acid identity with the amino acid sequence of SEQ IDNO: 56, at most 75% amino acid identity with the amino acid sequence ofSEQ ID NO: 56, at most 80% amino acid identity with the amino acidsequence of SEQ ID NO: 56, at most 85% amino acid identity with theamino acid sequence of SEQ ID NO: 56, at most 90% amino acid identitywith the amino acid sequence of SEQ ID NO: 56 or at most 95% amino acididentity with the amino acid sequence of SEQ ID NO: 56.

In other aspects of this embodiment, a LIF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to theamino acid sequence of SEQ ID NO: 56. In other aspects of thisembodiment, a LIF comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to the amino acid sequence of SEQ IDNO: 56. In yet other aspects of this embodiment, a LIF comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 56. In other aspectsof this embodiment, a LIF comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to the amino acid sequenceof SEQ ID NO: 56. In still other aspects of this embodiment, a LIFcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to the amino acid sequence of SEQ ID NO: 56. In otheraspects of this embodiment, a LIF comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to the amino acid sequenceof SEQ ID NO: 56.

In other aspects of this embodiment, a LIF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to the aminoacid sequence of SEQ ID NO: 56. In other aspects of this embodiment, aLIF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to the amino acid sequence of SEQ ID NO: 56. Inyet other aspects of this embodiment, a LIF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to the aminoacid sequence of SEQ ID NO: 56. In other aspects of this embodiment, aLIF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to the amino acid sequence of SEQ ID NO: 56. In stillother aspects of this embodiment, a LIF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to the amino acidsequence of SEQ ID NO: 56. In other aspects of this embodiment, a LIFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 56.

In an embodiment, a binding domain comprises a CT-1. In anotherembodiment, a binding domain comprises a CT-1 of SEQ ID NO: 57. Inanother embodiment, a binding domain is derived from a CT-1. In anotherembodiment, a binding domain is derived from a CT-1 of SEQ ID NO: 57. Inaspects of this embodiment, a CT-1 comprises a polypeptide having, e.g.,at least 70% amino acid identity with the amino acid sequence of SEQ IDNO: 57, at least 75% amino acid identity with the amino acid sequence ofSEQ ID NO: 57, at least 80% amino acid identity with the amino acidsequence of SEQ ID NO: 57, at least 85% amino acid identity with theamino acid sequence of SEQ ID NO: 57, at least 90% amino acid identitywith the amino acid sequence of SEQ ID NO: 57 or at least 95% amino acididentity with the amino acid sequence of SEQ ID NO: 57. In yet otheraspects of this embodiment, a CT-1 comprises a polypeptide having, e.g.,at most 70% amino acid identity with the amino acid sequence of SEQ IDNO: 57, at most 75% amino acid identity with the amino acid sequence ofSEQ ID NO: 57, at most 80% amino acid identity with the amino acidsequence of SEQ ID NO: 57, at most 85% amino acid identity with theamino acid sequence of SEQ ID NO: 57, at most 90% amino acid identitywith the amino acid sequence of SEQ ID NO: 57 or at most 95% amino acididentity with the amino acid sequence of SEQ ID NO: 57.

In other aspects of this embodiment, a CT-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to theamino acid sequence of SEQ ID NO: 57. In other aspects of thisembodiment, a CT-1 comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to the amino acid sequence of SEQ IDNO: 57. In yet other aspects of this embodiment, a CT-1 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 57. In other aspectsof this embodiment, a CT-1 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to the amino acid sequenceof SEQ ID NO: 57. In still other aspects of this embodiment, a CT-1comprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to the amino acid sequence of SEQ ID NO: 57. In otheraspects of this embodiment, a CT-1 comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to the amino acid sequenceof SEQ ID NO: 57.

In other aspects of this embodiment, a CT-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to the aminoacid sequence of SEQ ID NO: 57. In other aspects of this embodiment, aCT-1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to the amino acid sequence of SEQ ID NO: 57. Inyet other aspects of this embodiment, a CT-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to the aminoacid sequence of SEQ ID NO: 57. In other aspects of this embodiment, aCT-1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to the amino acid sequence of SEQ ID NO: 57. In stillother aspects of this embodiment, a CT-1 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to the amino acidsequence of SEQ ID NO: 57. In other aspects of this embodiment, a CT-1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 57.

In an embodiment, a binding domain comprises a CLC. In anotherembodiment, a binding domain comprises a CLC of SEQ ID NO: 58. Inanother embodiment, a binding domain is derived from a CLC. In anotherembodiment, a binding domain is derived from a CLC of SEQ ID NO: 58. Inaspects of this embodiment, a CLC comprises a polypeptide having, e.g.,at least 70% amino acid identity with the amino acid sequence of SEQ IDNO: 58, at least 75% amino acid identity with the amino acid sequence ofSEQ ID NO: 58, at least 80% amino acid identity with the amino acidsequence of SEQ ID NO: 58, at least 85% amino acid identity with theamino acid sequence of SEQ ID NO: 58, at least 90% amino acid identitywith the amino acid sequence of SEQ ID NO: 58 or at least 95% amino acididentity with the amino acid sequence of SEQ ID NO: 58. In yet otheraspects of this embodiment, a CLC comprises a polypeptide having, e.g.,at most 70% amino acid identity with the amino acid sequence of SEQ IDNO: 58, at most 75% amino acid identity with the amino acid sequence ofSEQ ID NO: 58, at most 80% amino acid identity with the amino acidsequence of SEQ ID NO: 58, at most 85% amino acid identity with theamino acid sequence of SEQ ID NO: 58, at most 90% amino acid identitywith the amino acid sequence of SEQ ID NO: 58 or at most 95% amino acididentity with the amino acid sequence of SEQ ID NO: 58.

In other aspects of this embodiment, a CLC comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to theamino acid sequence of SEQ ID NO: 58. In other aspects of thisembodiment, a CLC comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to the amino acid sequence of SEQ IDNO: 58. In yet other aspects of this embodiment, a CLC comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 58. In other aspectsof this embodiment, a CLC comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to the amino acid sequenceof SEQ ID NO: 58. In still other aspects of this embodiment, a CLCcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to the amino acid sequence of SEQ ID NO: 58. In otheraspects of this embodiment, a CLC comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to the amino acid sequenceof SEQ ID NO: 58.

In other aspects of this embodiment, a CLC comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to the aminoacid sequence of SEQ ID NO: 58. In other aspects of this embodiment, aCLC comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to the amino acid sequence of SEQ ID NO: 58. Inyet other aspects of this embodiment, a CLC comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to the aminoacid sequence of SEQ ID NO: 58. In other aspects of this embodiment, aCLC comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to the amino acid sequence of SEQ ID NO: 58. In stillother aspects of this embodiment, a CLC comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to the amino acidsequence of SEQ ID NO: 58. In other aspects of this embodiment, a CLCcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 58.

In an embodiment, a binding domain comprises a IL-1. In anotherembodiment, a binding domain comprises a IL-1 of SEQ ID NO: 59. Inanother embodiment, a binding domain is derived from an IL-1. In anotherembodiment, a binding domain is derived from an IL-1 of SEQ ID NO: 59.In an aspect of this embodiment, a binding domain is derived from anIL-1 comprising amino acids 123-265 of SEQ ID NO: 59.

In other aspects of this embodiment, an IL-1 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 123-265of SEQ ID NO: 59, at least 75% amino acid identity with amino acids123-265 of SEQ ID NO: 59, at least 80% amino acid identity with aminoacids 123-265 of SEQ ID NO: 59, at least 85% amino acid identity withamino acids 123-265 of SEQ ID NO: 59, at least 90% amino acid identitywith amino acids 123-265 of SEQ ID NO: 59 or at least 95% amino acididentity with amino acids 123-265 of SEQ ID NO: 59. In yet other aspectsof this embodiment, an IL-1 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 123-265 of SEQ ID NO: 59,at most 75% amino acid identity with amino acids 123-265 of SEQ ID NO:59, at most 80% amino acid identity with amino acids 123-265 of SEQ IDNO: 59, at most 85% amino acid identity with amino acids 123-265 of SEQID NO: 59, at most 90% amino acid identity with amino acids 123-265 ofSEQ ID NO: 59 or at most 95% amino acid identity with amino acids123-265 of SEQ ID NO: 59.

In other aspects of this embodiment, an IL-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 123-265 of SEQ ID NO: 59. In other aspects of this embodiment, anIL-1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 123-265 of SEQ ID NO: 59. In yetother aspects of this embodiment, an IL-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 123-265 of SEQ ID NO: 59. In other aspects of this embodiment, anIL-1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 123-265 of SEQ ID NO: 59. In stillother aspects of this embodiment, an IL-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 123-265 of SEQ ID NO: 59. In other aspects of this embodiment, anIL-1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 123-265 of SEQ ID NO: 59.

In other aspects of this embodiment, an IL-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 123-265 of SEQ ID NO: 59. In other aspects of this embodiment, anIL-1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 123-265 of SEQ ID NO: 59. In yetother aspects of this embodiment, an IL-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids123-265 of SEQ ID NO: 59.

In other aspects of this embodiment, an IL-1 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids123-265 of SEQ ID NO: 59. In still other aspects of this embodiment, anIL-1 comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 123-265 of SEQ ID NO: 59. In otheraspects of this embodiment, an IL-1 comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 123-265 ofSEQ ID NO: 59.

In an embodiment, a binding domain comprises a IL-2. In anotherembodiment, a binding domain comprises a IL-2 of SEQ ID NO: 60. Inanother embodiment, a binding domain is derived from an IL-2. In anotherembodiment, a binding domain is derived from an IL-2 of SEQ ID NO: 60.In an aspect of this embodiment, a binding domain is derived from anIL-2 comprising amino acids 21-153 of SEQ ID NO: 60.

In other aspects of this embodiment, an IL-2 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 21-153of SEQ ID NO: 60, at least 75% amino acid identity with amino acids21-153 of SEQ ID NO: 60, at least 80% amino acid identity with aminoacids 21-153 of SEQ ID NO: 60, at least 85% amino acid identity withamino acids 21-153 of SEQ ID NO: 60, at least 90% amino acid identitywith amino acids 21-153 of SEQ ID NO: 60 or at least 95% amino acididentity with amino acids 21-153 of SEQ ID NO: 60. In yet other aspectsof this embodiment, an IL-2 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 21-153 of SEQ ID NO: 60,at most 75% amino acid identity with amino acids 21-153 of SEQ ID NO:60, at most 80% amino acid identity with amino acids 21-153 of SEQ IDNO: 60, at most 85% amino acid identity with amino acids 21-153 of SEQID NO: 60, at most 90% amino acid identity with amino acids 21-153 ofSEQ ID NO: 60 or at most 95% amino acid identity with amino acids 21-153of SEQ ID NO: 60.

In other aspects of this embodiment, an IL-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 21-153 of SEQ ID NO: 60. In other aspects of this embodiment, anIL-2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 21-153 of SEQ ID NO: 60. In yetother aspects of this embodiment, an IL-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 21-153 of SEQ ID NO: 60. In other aspects of this embodiment, anIL-2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 21-153 of SEQ ID NO: 60. In stillother aspects of this embodiment, an IL-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 21-153 of SEQ ID NO: 60. In other aspects of this embodiment, anIL-2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 21-153 of SEQ ID NO: 60.

In other aspects of this embodiment, an IL-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 21-153 of SEQ ID NO: 60. In other aspects of this embodiment, anIL-2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 21-153 of SEQ ID NO: 60. In yetother aspects of this embodiment, an IL-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids21-153 of SEQ ID NO: 60. In other aspects of this embodiment, an IL-2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 21-153 of SEQ ID NO: 60. In still other aspectsof this embodiment, an IL-2 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 21-153 of SEQ IDNO: 60. In other aspects of this embodiment, an IL-2 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 21-153 of SEQ ID NO: 60.

In an embodiment, a binding domain comprises a IL-6. In anotherembodiment, a binding domain comprises a IL-6 of SEQ ID NO: 61. Inanother embodiment, a binding domain is derived from an IL-6. In anotherembodiment, a binding domain is derived from an IL-6 of SEQ ID NO: 61.In an aspect of this embodiment, a binding domain is derived from anIL-6 comprising amino acids 57-210 of SEQ ID NO: 61.

In other aspects of this embodiment, an IL-6 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 57-210of SEQ ID NO: 61, at least 75% amino acid identity with amino acids57-210 of SEQ ID NO: 61, at least 80% amino acid identity with aminoacids 57-210 of SEQ ID NO: 61, at least 85% amino acid identity withamino acids 57-210 of SEQ ID NO: 61, at least 90% amino acid identitywith amino acids 57-210 of SEQ ID NO: 61 or at least 95% amino acididentity with amino acids 57-210 of SEQ ID NO: 61. In yet other aspectsof this embodiment, an IL-6 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 57-210 of SEQ ID NO: 61,at most 75% amino acid identity with amino acids 57-210 of SEQ ID NO:61, at most 80% amino acid identity with amino acids 57-210 of SEQ IDNO: 61, at most 85% amino acid identity with amino acids 57-210 of SEQID NO: 61, at most 90% amino acid identity with amino acids 57-210 ofSEQ ID NO: 61 or at most 95% amino acid identity with amino acids 57-210of SEQ ID NO: 61.

In other aspects of this embodiment, an IL-6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 57-210 of SEQ ID NO: 61. In other aspects of this embodiment, anIL-6 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 57-210 of SEQ ID NO: 61. In yetother aspects of this embodiment, an IL-6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 57-210 of SEQ ID NO: 61. In other aspects of this embodiment, anIL-6 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 57-210 of SEQ ID NO: 61. In stillother aspects of this embodiment, an IL-6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 57-210 of SEQ ID NO: 61. In other aspects of this embodiment, anIL-6 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 57-210 of SEQ ID NO: 61.

In other aspects of this embodiment, an IL-6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 57-210 of SEQ ID NO: 61. In other aspects of this embodiment, anIL-6 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 57-210 of SEQ ID NO: 61. In yetother aspects of this embodiment, an IL-6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids57-210 of SEQ ID NO: 61. In other aspects of this embodiment, an IL-6comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 57-210 of SEQ ID NO: 61. In still other aspectsof this embodiment, an IL-6 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 57-210 of SEQ IDNO: 61. In other aspects of this embodiment, an IL-6 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 57-210 of SEQ ID NO: 61.

In an embodiment, a binding domain comprises a IL-8. In anotherembodiment, a binding domain comprises a IL-8 of SEQ ID NO: 62. Inanother embodiment, a binding domain is derived from an IL-8. In anotherembodiment, a binding domain is derived from an IL-8 of SEQ ID NO: 62.In an aspect of this embodiment, a binding domain is derived from anIL-8 comprising amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62.

In other aspects of this embodiment, an IL-8 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 21-99 oramino acids 31-94 of SEQ ID NO: 62, at least 75% amino acid identitywith amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62, at least80% amino acid identity with amino acids 21-99 or amino acids 31-94 ofSEQ ID NO: 62, at least 85% amino acid identity with amino acids 21-99or amino acids 31-94 of SEQ ID NO: 62, at least 90% amino acid identitywith amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62 or at least95% amino acid identity with amino acids 21-99 or amino acids 31-94 ofSEQ ID NO: 62. In yet other aspects of this embodiment, an IL-8comprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62, at most75% amino acid identity with amino acids 21-99 or amino acids 31-94 ofSEQ ID NO: 62, at most 80% amino acid identity with amino acids 21-99 oramino acids 31-94 of SEQ ID NO: 62, at most 85% amino acid identity withamino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62, at most 90%amino acid identity with amino acids 21-99 or amino acids 31-94 of SEQID NO: 62 or at most 95% amino acid identity with amino acids 21-99 oramino acids 31-94 of SEQ ID NO: 62.

In other aspects of this embodiment, an IL-8 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 21-99 or amino acids 31-94 of SEQ ID NO: 62. In other aspects ofthis embodiment, an IL-8 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 21-99 oramino acids 31-94 of SEQ ID NO: 62. In yet other aspects of thisembodiment, an IL-8 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 21-99 or amino acids 31-94of SEQ ID NO: 62. In other aspects of this embodiment, an IL-8 comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62. Instill other aspects of this embodiment, an IL-8 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 21-99 or amino acids 31-94 of SEQ ID NO: 62. In other aspects ofthis embodiment, an IL-8 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 21-99 oramino acids 31-94 of SEQ ID NO: 62.

In other aspects of this embodiment, an IL-8 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 21-99 or amino acids 31-94 of SEQ ID NO: 62. In other aspects ofthis embodiment, an IL-8 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 21-99 oramino acids 31-94 of SEQ ID NO: 62. In yet other aspects of thisembodiment, an IL-8 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 21-99 or amino acids 31-94of SEQ ID NO: 62. In other aspects of this embodiment, an IL-8 comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 21-99 or amino acids 31-94 of SEQ ID NO: 62. In still otheraspects of this embodiment, an IL-8 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 21-99 oramino acids 31-94 of SEQ ID NO: 62. In other aspects of this embodiment,an IL-8 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 21-99 or amino acids 31-94 of SEQ IDNO: 62.

In an embodiment, a binding domain comprises a IL-10. In anotherembodiment, a binding domain comprises a IL-10 of SEQ ID NO: 63. Inanother embodiment, a binding domain is derived from an IL-10. Inanother embodiment, a binding domain is derived from an IL-10 of SEQ IDNO: 63. In an aspect of this embodiment, a binding domain is derivedfrom an IL-10 comprising amino acids 37-173 or amino acids 19-178 of SEQID NO: 63.

In other aspects of this embodiment, an IL-10 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 37-173or amino acids 19-178 of SEQ ID NO: 63, at least 75% amino acid identitywith amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63, at least80% amino acid identity with amino acids 37-173 or amino acids 19-178 ofSEQ ID NO: 63, at least 85% amino acid identity with amino acids 37-173or amino acids 19-178 of SEQ ID NO: 63, at least 90% amino acid identitywith amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63 or atleast 95% amino acid identity with amino acids 37-173 or amino acids19-178 of SEQ ID NO: 63. In yet other aspects of this embodiment, anIL-10 comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63,at most 75% amino acid identity with amino acids 37-173 or amino acids19-178 of SEQ ID NO: 63, at most 80% amino acid identity with aminoacids 37-173 or amino acids 19-178 of SEQ ID NO: 63, at most 85% aminoacid identity with amino acids 37-173 or amino acids 19-178 of SEQ IDNO: 63, at most 90% amino acid identity with amino acids 37-173 or aminoacids 19-178 of SEQ ID NO: 63 or at most 95% amino acid identity withamino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63.

In other aspects of this embodiment, an IL-10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 37-173 or amino acids 19-178 of SEQ ID NO: 63. In other aspects ofthis embodiment, an IL-10 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 37-173or amino acids 19-178 of SEQ ID NO: 63. In yet other aspects of thisembodiment, an IL-10 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 37-173 or amino acids19-178 of SEQ ID NO: 63. In other aspects of this embodiment, an IL-10comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 37-173 or amino acids 19-178 of SEQ IDNO: 63. In still other aspects of this embodiment, an IL-10 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63.In other aspects of this embodiment, an IL-10 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 37-173 or amino acids 19-178 of SEQ ID NO: 63.

In other aspects of this embodiment, an IL-10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 37-173 or amino acids 19-178 of SEQ ID NO: 63. In other aspects ofthis embodiment, an IL-10 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 37-173 oramino acids 19-178 of SEQ ID NO: 63. In yet other aspects of thisembodiment, an IL-10 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 37-173 or amino acids19-178 of SEQ ID NO: 63. In other aspects of this embodiment, an IL-10comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63.In still other aspects of this embodiment, an IL-10 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 37-173 or amino acids 19-178 of SEQ ID NO: 63. In otheraspects of this embodiment, an IL-10 comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 37-173 oramino acids 19-178 of SEQ ID NO: 63.

In an embodiment, a binding domain comprises a neuroleukin. In anotherembodiment, a binding domain comprises a neuroleukin of SEQ ID NO: 64.In another embodiment, a binding domain is derived from a neuroleukin.In another embodiment, a binding domain is derived from a neuroleukin ofSEQ ID NO: 64. In aspects of this embodiment, a neuroleukin comprises apolypeptide having, e.g., at least 70% amino acid identity with theamino acid sequence of SEQ ID NO: 64, at least 75% amino acid identitywith the amino acid sequence of SEQ ID NO: 64, at least 80% amino acididentity with the amino acid sequence of SEQ ID NO: 64, at least 85%amino acid identity with the amino acid sequence of SEQ ID NO: 64, atleast 90% amino acid identity with the amino acid sequence of SEQ ID NO:64 or at least 95% amino acid identity with the amino acid sequence ofSEQ ID NO: 64. In yet other aspects of this embodiment, a neuroleukincomprises a polypeptide having, e.g., at most 70% amino acid identitywith the amino acid sequence of SEQ ID NO: 64, at most 75% amino acididentity with the amino acid sequence of SEQ ID NO: 64, at most 80%amino acid identity with the amino acid sequence of SEQ ID NO: 64, atmost 85% amino acid identity with the amino acid sequence of SEQ ID NO:64, at most 90% amino acid identity with the amino acid sequence of SEQID NO: 64 or at most 95% amino acid identity with the amino acidsequence of SEQ ID NO: 64.

In other aspects of this embodiment, a neuroleukin comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to the amino acid sequence of SEQ ID NO: 64. In other aspectsof this embodiment, a neuroleukin comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to the amino acidsequence of SEQ ID NO: 64. In yet other aspects of this embodiment, aneuroleukin comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to the amino acid sequence of SEQ ID NO:64. In other aspects of this embodiment, a neuroleukin comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 64. In still otheraspects of this embodiment, a neuroleukin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to the aminoacid sequence of SEQ ID NO: 64. In other aspects of this embodiment, aneuroleukin comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to the amino acid sequence of SEQ ID NO:64.

In other aspects of this embodiment, a neuroleukin comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to the amino acid sequence of SEQ ID NO: 64. In other aspectsof this embodiment, a neuroleukin comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to the amino acid sequenceof SEQ ID NO: 64. In yet other aspects of this embodiment, a neuroleukincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 64. In other aspectsof this embodiment, a neuroleukin comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to the amino acid sequence ofSEQ ID NO: 64. In still other aspects of this embodiment, a neuroleukincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 64. In other aspectsof this embodiment, a neuroleukin comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to the amino acid sequence ofSEQ ID NO: 64.

In an embodiment, a binding domain comprises a VEGF. In anotherembodiment, a binding domain comprises a VEGF of SEQ ID NO: 65. Inanother embodiment, a binding domain is derived from a VEGF. In anotherembodiment, a binding domain is derived from a VEGF of SEQ ID NO: 65. Inaspects of this embodiment, a VEGF comprises a polypeptide having, e.g.,at least 70% amino acid identity with the amino acid sequence of SEQ IDNO: 65, at least 75% amino acid identity with the amino acid sequence ofSEQ ID NO: 65, at least 80% amino acid identity with the amino acidsequence of SEQ ID NO: 65, at least 85% amino acid identity with theamino acid sequence of SEQ ID NO: 65, at least 90% amino acid identitywith the amino acid sequence of SEQ ID NO: 65 or at least 95% amino acididentity with the amino acid sequence of SEQ ID NO: 65. In yet otheraspects of this embodiment, a VEGF comprises a polypeptide having, e.g.,at most 70% amino acid identity with the amino acid sequence of SEQ IDNO: 65, at most 75% amino acid identity with the amino acid sequence ofSEQ ID NO: 65, at most 80% amino acid identity with the amino acidsequence of SEQ ID NO: 65, at most 85% amino acid identity with theamino acid sequence of SEQ ID NO: 65, at most 90% amino acid identitywith the amino acid sequence of SEQ ID NO: 65 or at most 95% amino acididentity with the amino acid sequence of SEQ ID NO: 65.

In other aspects of this embodiment, a VEGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to theamino acid sequence of SEQ ID NO: 65. In other aspects of thisembodiment, a VEGF comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to the amino acid sequence of SEQ IDNO: 65. In yet other aspects of this embodiment, a VEGF comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 65. In other aspectsof this embodiment, a VEGF comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to the amino acid sequenceof SEQ ID NO: 65. In still other aspects of this embodiment, a VEGFcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to the amino acid sequence of SEQ ID NO: 65. In otheraspects of this embodiment, a VEGF comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to the amino acid sequenceof SEQ ID NO: 65.

In other aspects of this embodiment, a VEGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to the aminoacid sequence of SEQ ID NO: 65. In other aspects of this embodiment, aVEGF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to the amino acid sequence of SEQ ID NO: 65. Inyet other aspects of this embodiment, a VEGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to the aminoacid sequence of SEQ ID NO: 65. In other aspects of this embodiment, aVEGF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to the amino acid sequence of SEQ ID NO: 65. In stillother aspects of this embodiment, a VEGF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to the amino acidsequence of SEQ ID NO: 65. In other aspects of this embodiment, a VEGFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 65.

In an embodiment, a binding domain comprises a IGF-1. In anotherembodiment, a binding domain comprises a IGF-1 of SEQ ID NO: 66. Inanother embodiment, a binding domain is derived from an IGF-1. Inanother embodiment, a binding domain is derived from an IGF-1 of SEQ IDNO: 66. In an aspect of this embodiment, a binding domain is derivedfrom an IGF-1 comprising amino acids 52-109 or amino acids 49-118 of SEQID NO: 66.

In other aspects of this embodiment, an IGF-1 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 52-109or amino acids 49-118 of SEQ ID NO: 66, at least 75% amino acid identitywith amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66, at least80% amino acid identity with amino acids 52-109 or amino acids 49-118 ofSEQ ID NO: 66, at least 85% amino acid identity with amino acids 52-109or amino acids 49-118 of SEQ ID NO: 66, at least 90% amino acid identitywith amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66 or atleast 95% amino acid identity with amino acids 52-109 or amino acids49-118 of SEQ ID NO: 66. In yet other aspects of this embodiment, anIGF-1 comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66,at most 75% amino acid identity with amino acids 52-109 or amino acids49-118 of SEQ ID NO: 66, at most 80% amino acid identity with aminoacids 52-109 or amino acids 49-118 of SEQ ID NO: 66, at most 85% aminoacid identity with amino acids 52-109 or amino acids 49-118 of SEQ IDNO: 66, at most 90% amino acid identity with amino acids 52-109 or aminoacids 49-118 of SEQ ID NO: 66 or at most 95% amino acid identity withamino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66.

In other aspects of this embodiment, an IGF-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 52-109 or amino acids 49-118 of SEQ ID NO: 66. In other aspects ofthis embodiment, an IGF-1 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 52-109or amino acids 49-118 of SEQ ID NO: 66. In yet other aspects of thisembodiment, an IGF-1 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 52-109 or amino acids49-118 of SEQ ID NO: 66. In other aspects of this embodiment, an IGF-1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 52-109 or amino acids 49-118 of SEQ IDNO: 66. In still other aspects of this embodiment, an IGF-1 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66.In other aspects of this embodiment, an IGF-1 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 52-109 or amino acids 49-118 of SEQ ID NO: 66.

In other aspects of this embodiment, an IGF-1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 52-109 or amino acids 49-118 of SEQ ID NO: 66. In other aspects ofthis embodiment, an IGF-1 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 52-109 oramino acids 49-118 of SEQ ID NO: 66. In yet other aspects of thisembodiment, an IGF-1 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 52-109 or amino acids49-118 of SEQ ID NO: 66. In other aspects of this embodiment, an IGF-1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66.In still other aspects of this embodiment, an IGF-1 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 52-109 or amino acids 49-118 of SEQ ID NO: 66. In otheraspects of this embodiment, an IGF-1 comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 52-109 oramino acids 49-118 of SEQ ID NO: 66.

In an embodiment, a binding domain comprises a IGF-2. In anotherembodiment, a binding domain comprises a IGF-2 of SEQ ID NO: 67. Inanother embodiment, a binding domain is derived from an IGF-2. Inanother embodiment, a binding domain is derived from an IGF-2 of SEQ IDNO: 67. In an aspect of this embodiment, a binding domain is derivedfrom an IGF-2 comprising amino acids 31-84 or amino acids 25-180 of SEQID NO: 67.

In other aspects of this embodiment, an IGF-2 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 31-84 oramino acids 25-180 of SEQ ID NO: 67, at least 75% amino acid identitywith amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67, at least80% amino acid identity with amino acids 31-84 or amino acids 25-180 ofSEQ ID NO: 67, at least 85% amino acid identity with amino acids 31-84or amino acids 25-180 of SEQ ID NO: 67, at least 90% amino acid identitywith amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67 or atleast 95% amino acid identity with amino acids 31-84 or amino acids25-180 of SEQ ID NO: 67. In yet other aspects of this embodiment, anIGF-2 comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67,at most 75% amino acid identity with amino acids 31-84 or amino acids25-180 of SEQ ID NO: 67, at most 80% amino acid identity with aminoacids 31-84 or amino acids 25-180 of SEQ ID NO: 67, at most 85% aminoacid identity with amino acids 31-84 or amino acids 25-180 of SEQ ID NO:67, at most 90% amino acid identity with amino acids 31-84 or aminoacids 25-180 of SEQ ID NO: 67 or at most 95% amino acid identity withamino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67.

In other aspects of this embodiment, an IGF-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 31-84 or amino acids 25-180 of SEQ ID NO: 67. In other aspects ofthis embodiment, an IGF-2 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 31-84 oramino acids 25-180 of SEQ ID NO: 67. In yet other aspects of thisembodiment, an IGF-2 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 31-84 or amino acids 25-180of SEQ ID NO: 67. In other aspects of this embodiment, an IGF-2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 31-84 or amino acids 25-180 of SEQ IDNO: 67. In still other aspects of this embodiment, an IGF-2 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67.

In other aspects of this embodiment, an IGF-2 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 31-84 or amino acids 25-180 of SEQ ID NO: 67.

In other aspects of this embodiment, an IGF-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 31-84 or amino acids 25-180 of SEQ ID NO: 67. In other aspects ofthis embodiment, an IGF-2 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 31-84 oramino acids 25-180 of SEQ ID NO: 67. In yet other aspects of thisembodiment, an IGF-2 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 31-84 or amino acids 25-180of SEQ ID NO: 67. In other aspects of this embodiment, an IGF-2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 31-84 or amino acids 25-180 of SEQ ID NO: 67. Instill other aspects of this embodiment, an IGF-2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids31-84 or amino acids 25-180 of SEQ ID NO: 67. In other aspects of thisembodiment, an IGF-2 comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 31-84 or amino acids 25-180of SEQ ID NO: 67.

In an embodiment, a binding domain comprises a EGF. In anotherembodiment, a binding domain comprises a EGF of SEQ ID NO: 68. Inanother embodiment, a binding domain is derived from an EGF. In anotherembodiment, a binding domain is derived from an EGF of SEQ ID NO: 68. Inaspects of this embodiment, an EGF comprises a polypeptide having, e.g.,at least 70% amino acid identity with the amino acid sequence of SEQ IDNO: 68, at least 75% amino acid identity with the amino acid sequence ofSEQ ID NO: 68, at least 80% amino acid identity with the amino acidsequence of SEQ ID NO: 68, at least 85% amino acid identity with theamino acid sequence of SEQ ID NO: 68, at least 90% amino acid identitywith the amino acid sequence of SEQ ID NO: 68 or at least 95% amino acididentity with the amino acid sequence of SEQ ID NO: 68. In yet otheraspects of this embodiment, an EGF comprises a polypeptide having, e.g.,at most 70% amino acid identity with the amino acid sequence of SEQ IDNO: 68, at most 75% amino acid identity with the amino acid sequence ofSEQ ID NO: 68, at most 80% amino acid identity with the amino acidsequence of SEQ ID NO: 68, at most 85% amino acid identity with theamino acid sequence of SEQ ID NO: 68, at most 90% amino acid identitywith the amino acid sequence of SEQ ID NO: 68 or at most 95% amino acididentity with the amino acid sequence of SEQ ID NO: 68.

In other aspects of this embodiment, an EGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to theamino acid sequence of SEQ ID NO: 68. In other aspects of thisembodiment, an EGF comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to the amino acid sequence of SEQ IDNO: 68. In yet other aspects of this embodiment, an EGF comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to the amino acid sequence of SEQ ID NO: 68. In other aspectsof this embodiment, an EGF comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to the amino acid sequenceof SEQ ID NO: 68. In still other aspects of this embodiment, an EGFcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to the amino acid sequence of SEQ ID NO: 68. In otheraspects of this embodiment, an EGF comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to the amino acid sequenceof SEQ ID NO: 68.

In other aspects of this embodiment, an EGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to the aminoacid sequence of SEQ ID NO: 68. In other aspects of this embodiment, anEGF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to the amino acid sequence of SEQ ID NO: 68. Inyet other aspects of this embodiment, an EGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to the aminoacid sequence of SEQ ID NO: 68. In other aspects of this embodiment, anEGF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to the amino acid sequence of SEQ ID NO: 68. In stillother aspects of this embodiment, an EGF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to the amino acidsequence of SEQ ID NO: 68. In other aspects of this embodiment, an EGFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to the amino acid sequence of SEQ ID NO: 68.

Another example of a binding domain disclosed in the presentspecification is, e.g., a neurotrophin, such as, e.g., a NGF, a BDNF, aNT-3 or a NT-5.

Thus, in an embodiment, a binding domain comprises a NGF. In anotherembodiment, a binding domain comprises a NGF of SEQ ID NO: 69. Inanother embodiment, a binding domain is derived from a NGF. In anotherembodiment, a binding domain is derived from a NGF of SEQ ID NO: 69. Inan aspect of this embodiment, a binding domain is derived from a NGFcomprising amino acids 139-257 of SEQ ID NO: 69.

In other aspects of this embodiment, a NGF comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 139-257of SEQ ID NO: 69, at least 75% amino acid identity with amino acids139-257 of SEQ ID NO: 69, at least 80% amino acid identity with aminoacids 139-257 of SEQ ID NO: 69, at least 85% amino acid identity withamino acids 139-257 of SEQ ID NO: 69, at least 90% amino acid identitywith amino acids 139-257 of SEQ ID NO: 69 or at least 95% amino acididentity with amino acids 139-257 of SEQ ID NO: 69. In yet other aspectsof this embodiment, a NGF comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 139-257 of SEQ ID NO: 69, atmost 75% amino acid identity with amino acids 139-257 of SEQ ID NO: 69,at most 80% amino acid identity with amino acids 139-257 of SEQ ID NO:69, at most 85% amino acid identity with amino acids 139-257 of SEQ IDNO: 69, at most 90% amino acid identity with amino acids 139-257 of SEQID NO: 69 or at most 95% amino acid identity with amino acids 139-257 ofSEQ ID NO: 69.

In other aspects of this embodiment, a NGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 139-257 of SEQ ID NO: 69. In other aspects of this embodiment, aNGF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 139-257 of SEQ ID NO: 69. In yetother aspects of this embodiment, a NGF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids139-257 of SEQ ID NO: 69. In other aspects of this embodiment, a NGFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 139-257 of SEQ ID NO: 69. In stillother aspects of this embodiment, a NGF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids139-257 of SEQ ID NO: 69. In other aspects of this embodiment, a NGFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 139-257 of SEQ ID NO: 69.

In other aspects of this embodiment, a NGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 139-257 of SEQ ID NO: 69. In other aspects of this embodiment, aNGF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 139-257 of SEQ ID NO: 69. In yetother aspects of this embodiment, a NGF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 139-257 ofSEQ ID NO: 69. In other aspects of this embodiment, a NGF comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 139-257 of SEQ ID NO: 69. In still other aspects of thisembodiment, a NGF comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 139-257 of SEQ ID NO: 69.In other aspects of this embodiment, a NGF comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids139-257 of SEQ ID NO: 69.

In an embodiment, a binding domain comprises a BDGF. In anotherembodiment, a binding domain comprises a BDGF of SEQ ID NO: 70. Inanother embodiment, a binding domain is derived from a BDGF. In anotherembodiment, a binding domain is derived from a BDGF of SEQ ID NO: 70. Inan aspect of this embodiment, a binding domain is derived from a BDGFcomprising amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70.

In other aspects of this embodiment, a BDGF comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 133-240or amino acids 129-247 of SEQ ID NO: 70, at least 75% amino acididentity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO:70, at least 80% amino acid identity with amino acids 133-240 or aminoacids 129-247 of SEQ ID NO: 70, at least 85% amino acid identity withamino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70, at least90% amino acid identity with amino acids 133-240 or amino acids 129-247of SEQ ID NO: 70 or at least 95% amino acid identity with amino acids133-240 or amino acids 129-247 of SEQ ID NO: 70. In yet other aspects ofthis embodiment, a BDGF comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 133-240 or amino acids 129-247of SEQ ID NO: 70, at most 75% amino acid identity with amino acids133-240 or amino acids 129-247 of SEQ ID NO: 70, at most 80% amino acididentity with amino acids 133-240 or amino acids 129-247 of SEQ ID NO:70, at most 85% amino acid identity with amino acids 133-240 or aminoacids 129-247 of SEQ ID NO: 70, at most 90% amino acid identity withamino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70 or at most95% amino acid identity with amino acids 133-240 or amino acids 129-247of SEQ ID NO: 70.

In other aspects of this embodiment, a BDGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 133-240 or amino acids 129-247 of SEQ ID NO: 70. In other aspectsof this embodiment, a BDGF comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 133-240or amino acids 129-247 of SEQ ID NO: 70. In yet other aspects of thisembodiment, a BDGF comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 133-240 or amino acids129-247 of SEQ ID NO: 70. In other aspects of this embodiment, a BDGFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 133-240 or amino acids 129-247 of SEQID NO: 70. In still other aspects of this embodiment, a BDGF comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70.In other aspects of this embodiment, a BDGF comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 133-240 or amino acids 129-247 of SEQ ID NO: 70.

In other aspects of this embodiment, a BDGF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 133-240 or amino acids 129-247 of SEQ ID NO: 70. In other aspectsof this embodiment, a BDGF comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 133-240 oramino acids 129-247 of SEQ ID NO: 70. In yet other aspects of thisembodiment, a BDGF comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 133-240 or amino acids129-247 of SEQ ID NO: 70. In other aspects of this embodiment, a BDGFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70.In still other aspects of this embodiment, a BDGF comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 133-240 or amino acids 129-247 of SEQ ID NO: 70. In otheraspects of this embodiment, a BDGF comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 133-240 or aminoacids 129-247 of SEQ ID NO: 70.

In an embodiment, a binding domain comprises a NT-3. In anotherembodiment, a binding domain comprises a NT-3 of SEQ ID NO: 71. Inanother embodiment, a binding domain is derived from a NT-3. In anotherembodiment, a binding domain is derived from a NT-3 of SEQ ID NO: 71. Inan aspect of this embodiment, a binding domain is derived from a NT-3comprising amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71.

In other aspects of this embodiment, a NT-3 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 144-249or amino acids 19-257 of SEQ ID NO: 71, at least 75% amino acid identitywith amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71, atleast 80% amino acid identity with amino acids 144-249 or amino acids19-257 of SEQ ID NO: 71, at least 85% amino acid identity with aminoacids 144-249 or amino acids 19-257 of SEQ ID NO: 71, at least 90% aminoacid identity with amino acids 144-249 or amino acids 19-257 of SEQ IDNO: 71 or at least 95% amino acid identity with amino acids 144-249 oramino acids 19-257 of SEQ ID NO: 71. In yet other aspects of thisembodiment, a NT-3 comprises a polypeptide having, e.g., at most 70%amino acid identity with amino acids 144-249 or amino acids 19-257 ofSEQ ID NO: 71, at most 75% amino acid identity with amino acids 144-249or amino acids 19-257 of SEQ ID NO: 71, at most 80% amino acid identitywith amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71, at most85% amino acid identity with amino acids 144-249 or amino acids 19-257of SEQ ID NO: 71, at most 90% amino acid identity with amino acids144-249 or amino acids 19-257 of SEQ ID NO: 71 or at most 95% amino acididentity with amino acids 144-249 or amino acids 19-257 of SEQ ID NO:71.

In other aspects of this embodiment, a NT-3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 144-249 or amino acids 19-257 of SEQ ID NO: 71. In other aspectsof this embodiment, a NT-3 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 144-249or amino acids 19-257 of SEQ ID NO: 71. In yet other aspects of thisembodiment, a NT-3 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 144-249 or amino acids19-257 of SEQ ID NO: 71. In other aspects of this embodiment, a NT-3comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 144-249 or amino acids 19-257 of SEQID NO: 71. In still other aspects of this embodiment, a NT-3 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71.In other aspects of this embodiment, a NT-3 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 144-249 or amino acids 19-257 of SEQ ID NO: 71.

In other aspects of this embodiment, a NT-3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 144-249 or amino acids 19-257 of SEQ ID NO: 71. In other aspectsof this embodiment, a NT-3 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 144-249 oramino acids 19-257 of SEQ ID NO: 71. In yet other aspects of thisembodiment, a NT-3 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 144-249 or amino acids19-257 of SEQ ID NO: 71. In other aspects of this embodiment, a NT-3comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71.In still other aspects of this embodiment, a NT-3 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 144-249 or amino acids 19-257 of SEQ ID NO: 71. In otheraspects of this embodiment, a NT-3 comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 144-249 or aminoacids 19-257 of SEQ ID NO: 71.

In an embodiment, a binding domain comprises a NT-4/5. In anotherembodiment, a binding domain comprises a NT-4/5 of SEQ ID NO: 72. Inanother embodiment, a binding domain is derived from a NT-4/5. Inanother embodiment, a binding domain is derived from a NT-4/5 of SEQ IDNO: 72. In an aspect of this embodiment, a binding domain is derivedfrom a NT-4/5 comprising amino acids 89-202 or amino acids 81-210 of SEQID NO: 72.

In other aspects of this embodiment, a NT-4/5 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 89-202or amino acids 81-210 of SEQ ID NO: 72, at least 75% amino acid identitywith amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72, at least80% amino acid identity with amino acids 89-202 or amino acids 81-210 ofSEQ ID NO: 72, at least 85% amino acid identity with amino acids 89-202or amino acids 81-210 of SEQ ID NO: 72, at least 90% amino acid identitywith amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72 or atleast 95% amino acid identity with amino acids 89-202 or amino acids81-210 of SEQ ID NO: 72. In yet other aspects of this embodiment, aNT-4/5 comprises a polypeptide having, e.g., at most 70% amino acididentity with amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72,at most 75% amino acid identity with amino acids 89-202 or amino acids81-210 of SEQ ID NO: 72, at most 80% amino acid identity with aminoacids 89-202 or amino acids 81-210 of SEQ ID NO: 72, at most 85% aminoacid identity with amino acids 89-202 or amino acids 81-210 of SEQ IDNO: 72, at most 90% amino acid identity with amino acids 89-202 or aminoacids 81-210 of SEQ ID NO: 72 or at most 95% amino acid identity withamino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72.

In other aspects of this embodiment, a NT-4/5 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 89-202 or amino acids 81-210 of SEQ ID NO: 72. In other aspects ofthis embodiment, a NT-4/5 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 89-202or amino acids 81-210 of SEQ ID NO: 72. In yet other aspects of thisembodiment, a NT-4/5 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 89-202 or amino acids81-210 of SEQ ID NO: 72. In other aspects of this embodiment, a NT-4/5comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 89-202 or amino acids 81-210 of SEQ IDNO: 72. In still other aspects of this embodiment, a NT-4/5 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72.In other aspects of this embodiment, a NT-4/5 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 89-202 or amino acids 81-210 of SEQ ID NO: 72.

In other aspects of this embodiment, a NT-4/5 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 89-202 or amino acids 81-210 of SEQ ID NO: 72. In other aspects ofthis embodiment, a NT-4/5 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 89-202 oramino acids 81-210 of SEQ ID NO: 72. In yet other aspects of thisembodiment, a NT-4/5 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 89-202 or amino acids81-210 of SEQ ID NO: 72. In other aspects of this embodiment, a NT-4/5comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72.In still other aspects of this embodiment, a NT-4/5 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 89-202 or amino acids 81-210 of SEQ ID NO: 72. In otheraspects of this embodiment, a NT-4/5 comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 89-202 oramino acids 81-210 of SEQ ID NO: 72.

Another example of a binding domain disclosed in the presentspecification is, e.g., a GDNF, a neurturin, a persephrin or an artemin.

Thus, in an embodiment, a binding domain comprises a GDNF. In anotherembodiment, a binding domain comprises a GDNF of SEQ ID NO: 73. Inanother embodiment, a binding domain is derived from a GDNF. In anotherembodiment, a binding domain is derived from a GDNF of SEQ ID NO: 73. Inan aspect of this embodiment, a binding domain is derived from a GDNFcomprising amino acids 118-211 of SEQ ID NO: 73.

In other aspects of this embodiment, a GDNF comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 118-211of SEQ ID NO: 73, at least 75% amino acid identity with amino acids118-211 of SEQ ID NO: 73, at least 80% amino acid identity with aminoacids 118-211 of SEQ ID NO: 73, at least 85% amino acid identity withamino acids 118-211 of SEQ ID NO: 73, at least 90% amino acid identitywith amino acids 118-211 of SEQ ID NO: 73 or at least 95% amino acididentity with amino acids 118-211 of SEQ ID NO: 73. In yet other aspectsof this embodiment, a GDNF comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 118-211 of SEQ ID NO: 73, atmost 75% amino acid identity with amino acids 118-211 of SEQ ID NO: 73,at most 80% amino acid identity with amino acids 118-211 of SEQ ID NO:73, at most 85% amino acid identity with amino acids 118-211 of SEQ IDNO: 73, at most 90% amino acid identity with amino acids 118-211 of SEQID NO: 73 or at most 95% amino acid identity with amino acids 118-211 ofSEQ ID NO: 73.

In other aspects of this embodiment, a GDNF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 118-211 of SEQ ID NO: 73. In other aspects of this embodiment, aGDNF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 118-211 of SEQ ID NO: 73. In yetother aspects of this embodiment, a GDNF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids118-211 of SEQ ID NO: 73. In other aspects of this embodiment, a GDNFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 118-211 of SEQ ID NO: 73. In stillother aspects of this embodiment, a GDNF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids118-211 of SEQ ID NO: 73. In other aspects of this embodiment, a GDNFcomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 118-211 of SEQ ID NO: 73.

In other aspects of this embodiment, a GDNF comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 118-211 of SEQ ID NO: 73. In other aspects of this embodiment, aGDNF comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 118-211 of SEQ ID NO: 73. In yetother aspects of this embodiment, a GDNF comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 118-211 ofSEQ ID NO: 73. In other aspects of this embodiment, a GDNF comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 118-211 of SEQ ID NO: 73. In still other aspects of thisembodiment, a GDNF comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 118-211 of SEQ ID NO: 73.In other aspects of this embodiment, a GDNF comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids118-211 of SEQ ID NO: 73.

In an embodiment, a binding domain comprises a Neurturin. In anotherembodiment, a binding domain comprises a Neurturin of SEQ ID NO: 74. Inanother embodiment, a binding domain is derived from a Neurturin. Inanother embodiment, a binding domain is derived from a Neurturin of SEQID NO: 74. In an aspect of this embodiment, a binding domain is derivedfrom a Neurturin comprising amino acids 107-196 or amino acids 96-197 ofSEQ ID NO: 74.

In other aspects of this embodiment, a Neurturin comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 107-196or amino acids 96-197 of SEQ ID NO: 74, at least 75% amino acid identitywith amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74, atleast 80% amino acid identity with amino acids 107-196 or amino acids96-197 of SEQ ID NO: 74, at least 85% amino acid identity with aminoacids 107-196 or amino acids 96-197 of SEQ ID NO: 74, at least 90% aminoacid identity with amino acids 107-196 or amino acids 96-197 of SEQ IDNO: 74 or at least 95% amino acid identity with amino acids 107-196 oramino acids 96-197 of SEQ ID NO: 74. In yet other aspects of thisembodiment, a Neurturin comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 107-196 or amino acids 96-197of SEQ ID NO: 74, at most 75% amino acid identity with amino acids107-196 or amino acids 96-197 of SEQ ID NO: 74, at most 80% amino acididentity with amino acids 107-196 or amino acids 96-197 of SEQ ID NO:74, at most 85% amino acid identity with amino acids 107-196 or aminoacids 96-197 of SEQ ID NO: 74, at most 90% amino acid identity withamino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74 or at most95% amino acid identity with amino acids 107-196 or amino acids 96-197of SEQ ID NO: 74.

In other aspects of this embodiment, a Neurturin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 107-196 or amino acids 96-197 of SEQ ID NO: 74. In other aspectsof this embodiment, a Neurturin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 107-196or amino acids 96-197 of SEQ ID NO: 74. In yet other aspects of thisembodiment, a Neurturin comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 107-196 oramino acids 96-197 of SEQ ID NO: 74. In other aspects of thisembodiment, a Neurturin comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 107-196 oramino acids 96-197 of SEQ ID NO: 74. In still other aspects of thisembodiment, a Neurturin comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 107-196 oramino acids 96-197 of SEQ ID NO: 74. In other aspects of thisembodiment, a Neurturin comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 107-196 oramino acids 96-197 of SEQ ID NO: 74.

In other aspects of this embodiment, a Neurturin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 107-196 or amino acids 96-197 of SEQ ID NO: 74. In other aspectsof this embodiment, a Neurturin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 107-196 oramino acids 96-197 of SEQ ID NO: 74. In yet other aspects of thisembodiment, a Neurturin comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 107-196 or aminoacids 96-197 of SEQ ID NO: 74. In other aspects of this embodiment, aNeurturin comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid deletions relative to amino acids 107-196 or amino acids 96-197 ofSEQ ID NO: 74. In still other aspects of this embodiment, a Neurturincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 107-196 or amino acids 96-197 of SEQ ID NO: 74.In other aspects of this embodiment, a Neurturin comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids107-196 or amino acids 96-197 of SEQ ID NO: 74.

In an embodiment, a binding domain comprises a Persephrin. In anotherembodiment, a binding domain comprises a Persephrin of SEQ ID NO: 75. Inanother embodiment, a binding domain is derived from a Persephrin. Inanother embodiment, a binding domain is derived from a Persephrin of SEQID NO: 75. In an aspect of this embodiment, a binding domain is derivedfrom a Persephrin comprising amino acids 66-155 of SEQ ID NO: 75.

In other aspects of this embodiment, a Persephrin comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 66-155 of SEQ ID NO: 75, at least 75% amino acid identity withamino acids 66-155 of SEQ ID NO: 75, at least 80% amino acid identitywith amino acids 66-155 of SEQ ID NO: 75, at least 85% amino acididentity with amino acids 66-155 of SEQ ID NO: 75, at least 90% aminoacid identity with amino acids 66-155 of SEQ ID NO: 75 or at least 95%amino acid identity with amino acids 66-155 of SEQ ID NO: 75. In yetother aspects of this embodiment, a Persephrin comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 66-155 ofSEQ ID NO: 75, at most 75% amino acid identity with amino acids 66-155of SEQ ID NO: 75, at most 80% amino acid identity with amino acids66-155 of SEQ ID NO: 75, at most 85% amino acid identity with aminoacids 66-155 of SEQ ID NO: 75, at most 90% amino acid identity withamino acids 66-155 of SEQ ID NO: 75 or at most 95% amino acid identitywith amino acids 66-155 of SEQ ID NO: 75.

In other aspects of this embodiment, a Persephrin comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 66-155 of SEQ ID NO: 75. In other aspects ofthis embodiment, a Persephrin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 66-155of SEQ ID NO: 75. In yet other aspects of this embodiment, a Persephrincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 66-155 of SEQ ID NO: 75. In otheraspects of this embodiment, a Persephrin comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids 66-155of SEQ ID NO: 75. In still other aspects of this embodiment, aPersephrin comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 66-155 of SEQ ID NO: 75. Inother aspects of this embodiment, a Persephrin comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 66-155 of SEQ ID NO: 75.

In other aspects of this embodiment, a Persephrin comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 66-155 of SEQ ID NO: 75. In other aspects ofthis embodiment, a Persephrin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 66-155 ofSEQ ID NO: 75. In yet other aspects of this embodiment, a Persephrincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 66-155 of SEQ ID NO: 75. In other aspects ofthis embodiment, a Persephrin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 66-155 of SEQ IDNO: 75. In still other aspects of this embodiment, a Persephrincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 66-155 of SEQ ID NO: 75. In other aspects ofthis embodiment, a Persephrin comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 66-155 of SEQ IDNO: 75.

In an embodiment, a binding domain comprises an Artemin. In anotherembodiment, a binding domain comprises an Artemin of SEQ ID NO: 76. Inanother embodiment, a binding domain is derived from an Artemin. Inanother embodiment, a binding domain is derived from an Artemin of SEQID NO: 76. In an aspect of this embodiment, a binding domain is derivedfrom an Artemin comprising amino acids 123-218 of SEQ ID NO: 76.

In other aspects of this embodiment, an Artemin comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 123-218of SEQ ID NO: 76, at least 75% amino acid identity with amino acids123-218 of SEQ ID NO: 76, at least 80% amino acid identity with aminoacids 123-218 of SEQ ID NO: 76, at least 85% amino acid identity withamino acids 123-218 of SEQ ID NO: 76, at least 90% amino acid identitywith amino acids 123-218 of SEQ ID NO: 76 or at least 95% amino acididentity with amino acids 123-218 of SEQ ID NO: 76. In yet other aspectsof this embodiment, an Artemin comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 123-218 of SEQ ID NO: 76,at most 75% amino acid identity with amino acids 123-218 of SEQ ID NO:76, at most 80% amino acid identity with amino acids 123-218 of SEQ IDNO: 76, at most 85% amino acid identity with amino acids 123-218 of SEQID NO: 76, at most 90% amino acid identity with amino acids 123-218 ofSEQ ID NO: 76 or at most 95% amino acid identity with amino acids123-218 of SEQ ID NO: 76.

In other aspects of this embodiment, an Artemin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 123-218 of SEQ ID NO: 76. In other aspects of this embodiment, anArtemin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 123-218 of SEQ ID NO: 76. In yetother aspects of this embodiment, an Artemin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 123-218 of SEQ ID NO: 76. In other aspects of this embodiment, anArtemin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 123-218 of SEQ ID NO: 76. In stillother aspects of this embodiment, an Artemin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 123-218 of SEQ ID NO: 76. In other aspects of this embodiment, anArtemin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 123-218 of SEQ ID NO: 76.

In other aspects of this embodiment, an Artemin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 123-218 of SEQ ID NO: 76. In other aspects of this embodiment, anArtemin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 123-218 of SEQ ID NO: 76. In yetother aspects of this embodiment, an Artemin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids123-218 of SEQ ID NO: 76. In other aspects of this embodiment, anArtemin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 123-218 of SEQ ID NO: 76. In stillother aspects of this embodiment, an Artemin comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids123-218 of SEQ ID NO: 76. In other aspects of this embodiment, anArtemin comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 123-218 of SEQ ID NO: 76.

Another example of a binding domain disclosed in the presentspecification is, e.g., a TGFβs, such as, e.g., TGFβ1, TGFβ2, TGFβ3 orTGFβ4.

Thus, in an embodiment, a binding domain comprises a TGFβ1. In anotherembodiment, a binding domain comprises a TGFβ1 of SEQ ID NO: 77. Inanother embodiment, a binding domain is derived from a TGFβ1. In anotherembodiment, a binding domain is derived from a TGFβ1 of SEQ ID NO: 77.In an aspect of this embodiment, a binding domain is derived from aTGFβ1 comprising amino acids 293-390 of SEQ ID NO: 77.

In other aspects of this embodiment, a TGFβ1 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 293-390of SEQ ID NO: 77, at least 75% amino acid identity with amino acids293-390 of SEQ ID NO: 77, at least 80% amino acid identity with aminoacids 293-390 of SEQ ID NO: 77, at least 85% amino acid identity withamino acids 293-390 of SEQ ID NO: 77, at least 90% amino acid identitywith amino acids 293-390 of SEQ ID NO: 77 or at least 95% amino acididentity with amino acids 293-390 of SEQ ID NO: 77. In yet other aspectsof this embodiment, a TGFβ1 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 293-390 of SEQ ID NO: 77,at most 75% amino acid identity with amino acids 293-390 of SEQ ID NO:77, at most 80% amino acid identity with amino acids 293-390 of SEQ IDNO: 77, at most 85% amino acid identity with amino acids 293-390 of SEQID NO: 77, at most 90% amino acid identity with amino acids 293-390 ofSEQ ID NO: 77 or at most 95% amino acid identity with amino acids293-390 of SEQ ID NO: 77.

In other aspects of this embodiment, a TGFβ1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 293-390 of SEQ ID NO: 77. In other aspects of this embodiment, aTGFβ1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 293-390 of SEQ ID NO: 77. In yetother aspects of this embodiment, a TGFβ1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 293-390 of SEQ ID NO: 77. In other aspects of this embodiment, aTGFβ1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 293-390 of SEQ ID NO: 77. In stillother aspects of this embodiment, a TGFβ1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 293-390 of SEQ ID NO: 77. In other aspects of this embodiment, aTGFβ1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 293-390 of SEQ ID NO: 77.

In other aspects of this embodiment, a TGFβ1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 293-390 of SEQ ID NO: 77. In other aspects of this embodiment, aTGFβ1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 293-390 of SEQ ID NO: 77. In yetother aspects of this embodiment, a TGFβ1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids293-390 of SEQ ID NO: 77. In other aspects of this embodiment, a TGFβ1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 293-390 of SEQ ID NO: 77. In still other aspectsof this embodiment, a TGFβ1 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 293-390 of SEQID NO: 77. In other aspects of this embodiment, a TGFβ1 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 293-390 of SEQ ID NO: 77.

In an embodiment, a binding domain comprises a TGFβ2. In anotherembodiment, a binding domain comprises a TGFβ2 of SEQ ID NO: 78. Inanother embodiment, a binding domain is derived from a TGFβ2. In anotherembodiment, a binding domain is derived from a TGFβ2 of SEQ ID NO: 77.In an aspect of this embodiment, a binding domain is derived from aTGFβ2 comprising amino acids 317-414 of SEQ ID NO: 78.

In other aspects of this embodiment, a TGFβ2 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 317-414of SEQ ID NO: 78, at least 75% amino acid identity with amino acids317-414 of SEQ ID NO: 78, at least 80% amino acid identity with aminoacids 317-414 of SEQ ID NO: 78, at least 85% amino acid identity withamino acids 317-414 of SEQ ID NO: 78, at least 90% amino acid identitywith amino acids 317-414 of SEQ ID NO: 78 or at least 95% amino acididentity with amino acids 317-414 of SEQ ID NO: 78. In yet other aspectsof this embodiment, a TGFβ2 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 317-414 of SEQ ID NO: 78,at most 75% amino acid identity with amino acids 317-414 of SEQ ID NO:78, at most 80% amino acid identity with amino acids 317-414 of SEQ IDNO: 78, at most 85% amino acid identity with amino acids 317-414 of SEQID NO: 78, at most 90% amino acid identity with amino acids 317-414 ofSEQ ID NO: 78 or at most 95% amino acid identity with amino acids317-414 of SEQ ID NO: 78.

In other aspects of this embodiment, a TGFβ2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 317-414 of SEQ ID NO: 78. In other aspects of this embodiment, aTGFβ2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 317-414 of SEQ ID NO: 78. In yetother aspects of this embodiment, a TGFβ2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 317-414 of SEQ ID NO: 78. In other aspects of this embodiment, aTGFβ2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 317-414 of SEQ ID NO: 78. In stillother aspects of this embodiment, a TGFβ2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 317-414 of SEQ ID NO: 78. In other aspects of this embodiment, aTGFβ2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 317-414 of SEQ ID NO: 78.

In other aspects of this embodiment, a TGFβ2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 317-414 of SEQ ID NO: 78. In other aspects of this embodiment, aTGFβ2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 317-414 of SEQ ID NO: 78. In yetother aspects of this embodiment, a TGFβ2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids317-414 of SEQ ID NO: 78. In other aspects of this embodiment, a TGFβ2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 317-414 of SEQ ID NO: 78. In still other aspectsof this embodiment, a TGFβ2 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 317-414 of SEQID NO: 78. In other aspects of this embodiment, a TGFβ2 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 317-414 of SEQ ID NO: 78.

In an embodiment, a binding domain comprises a TGFβ3. In anotherembodiment, a binding domain comprises a TGFβ3 of SEQ ID NO: 79. Inanother embodiment, a binding domain is derived from a TGFβ3. In anotherembodiment, a binding domain is derived from a TGFβ3 of SEQ ID NO: 77.In an aspect of this embodiment, a binding domain is derived from aTGFβ3 comprising amino acids 315-412 of SEQ ID NO: 79.

In other aspects of this embodiment, a TGFβ3 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 315-412of SEQ ID NO: 79, at least 75% amino acid identity with amino acids315-412 of SEQ ID NO: 79, at least 80% amino acid identity with aminoacids 315-412 of SEQ ID NO: 79, at least 85% amino acid identity withamino acids 315-412 of SEQ ID NO: 79, at least 90% amino acid identitywith amino acids 315-412 of SEQ ID NO: 79 or at least 95% amino acididentity with amino acids 315-412 of SEQ ID NO: 79. In yet other aspectsof this embodiment, a TGFβ3 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 315-412 of SEQ ID NO: 79,at most 75% amino acid identity with amino acids 315-412 of SEQ ID NO:79, at most 80% amino acid identity with amino acids 315-412 of SEQ IDNO: 79, at most 85% amino acid identity with amino acids 315-412 of SEQID NO: 79, at most 90% amino acid identity with amino acids 315-412 ofSEQ ID NO: 79 or at most 95% amino acid identity with amino acids315-412 of SEQ ID NO: 79.

In other aspects of this embodiment, a TGFβ3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 315-412 of SEQ ID NO: 79. In other aspects of this embodiment, aTGFβ3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 315-412 of SEQ ID NO: 79. In yetother aspects of this embodiment, a TGFβ3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 315-412 of SEQ ID NO: 79. In other aspects of this embodiment, aTGFβ3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 315-412 of SEQ ID NO: 79. In stillother aspects of this embodiment, a TGFβ3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 315-412 of SEQ ID NO: 79. In other aspects of this embodiment, aTGFβ3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 315-412 of SEQ ID NO: 79.

In other aspects of this embodiment, a TGFβ3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 315-412 of SEQ ID NO: 79. In other aspects of this embodiment, aTGFβ3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 315-412 of SEQ ID NO: 79. In yetother aspects of this embodiment, a TGFβ3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids315-412 of SEQ ID NO: 79. In other aspects of this embodiment, a TGFβ3comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 315-412 of SEQ ID NO: 79. In still other aspectsof this embodiment, a TGFβ3 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 315-412 of SEQID NO: 79. In other aspects of this embodiment, a TGFβ3 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 315-412 of SEQ ID NO: 79.

In an embodiment, a binding domain comprises a TGFβ4. In anotherembodiment, a binding domain comprises a TGFβ4 of SEQ ID NO: 80. Inanother embodiment, a binding domain is derived from a TGFβ4. In anotherembodiment, a binding domain is derived from a TGFβ4 of SEQ ID NO: 77.In an aspect of this embodiment, a binding domain is derived from aTGFβ4 comprising amino acids 276-373 of SEQ ID NO: 80.

In other aspects of this embodiment, a TGFβ4 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 276-373of SEQ ID NO: 80, at least 75% amino acid identity with amino acids276-373 of SEQ ID NO: 80, at least 80% amino acid identity with aminoacids 276-373 of SEQ ID NO: 80, at least 85% amino acid identity withamino acids 276-373 of SEQ ID NO: 80, at least 90% amino acid identitywith amino acids 276-373 of SEQ ID NO: 80 or at least 95% amino acididentity with amino acids 276-373 of SEQ ID NO: 80. In yet other aspectsof this embodiment, a TGFβ4 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 276-373 of SEQ ID NO: 80,at most 75% amino acid identity with amino acids 276-373 of SEQ ID NO:80, at most 80% amino acid identity with amino acids 276-373 of SEQ IDNO: 80, at most 85% amino acid identity with amino acids 276-373 of SEQID NO: 80, at most 90% amino acid identity with amino acids 276-373 ofSEQ ID NO: 80 or at most 95% amino acid identity with amino acids276-373 of SEQ ID NO: 80.

In other aspects of this embodiment, a TGFβ4 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 276-373 of SEQ ID NO: 80. In other aspects of this embodiment, aTGFβ4 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 276-373 of SEQ ID NO: 80. In yetother aspects of this embodiment, a TGFβ4 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 276-373 of SEQ ID NO: 80. In other aspects of this embodiment, aTGFβ4 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 276-373 of SEQ ID NO: 80. In stillother aspects of this embodiment, a TGFβ4 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 276-373 of SEQ ID NO: 80. In other aspects of this embodiment, aTGFβ4 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 276-373 of SEQ ID NO: 80.

In other aspects of this embodiment, a TGFβ4 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 276-373 of SEQ ID NO: 80. In other aspects of this embodiment, aTGFβ4 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 276-373 of SEQ ID NO: 80. In yetother aspects of this embodiment, a TGFβ4 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids276-373 of SEQ ID NO: 80. In other aspects of this embodiment, a TGFβ4comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 276-373 of SEQ ID NO: 80. In still other aspectsof this embodiment, a TGFβ4 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 276-373 of SEQID NO: 80. In other aspects of this embodiment, a TGFβ4 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 276-373 of SEQ ID NO: 80.

Another example of a binding domain disclosed in the presentspecification is, e.g., a BMPs, such as, e.g., BMP2, BMP3, BMP4, BMP5,BMP6, BMP7, BMP8 or BMP10.

Thus, in an embodiment, a binding domain comprises a BMP2. In anotherembodiment, a binding domain comprises a BMP2 of SEQ ID NO: 81. Inanother embodiment, a binding domain is derived from a BMP2. In anotherembodiment, a binding domain is derived from a BMP2 of SEQ ID NO: 81. Inan aspect of this embodiment, a binding domain is derived from a BMP2comprising amino acids 296-396 of SEQ ID NO: 81.

In other aspects of this embodiment, a BMP2 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 296-396of SEQ ID NO: 81, at least 75% amino acid identity with amino acids296-396 of SEQ ID NO: 81, at least 80% amino acid identity with aminoacids 296-396 of SEQ ID NO: 81, at least 85% amino acid identity withamino acids 296-396 of SEQ ID NO: 81, at least 90% amino acid identitywith amino acids 296-396 of SEQ ID NO: 81 or at least 95% amino acididentity with amino acids 296-396 of SEQ ID NO: 81. In yet other aspectsof this embodiment, a BMP2 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 296-396 of SEQ ID NO: 81, atmost 75% amino acid identity with amino acids 296-396 of SEQ ID NO: 81,at most 80% amino acid identity with amino acids 296-396 of SEQ ID NO:81, at most 85% amino acid identity with amino acids 296-396 of SEQ IDNO: 81, at most 90% amino acid identity with amino acids 296-396 of SEQID NO: 81 or at most 95% amino acid identity with amino acids 296-396 ofSEQ ID NO: 81.

In other aspects of this embodiment, a BMP2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 296-396 of SEQ ID NO: 81. In other aspects of this embodiment, aBMP2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 296-396 of SEQ ID NO: 81. In yetother aspects of this embodiment, a BMP2 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids296-396 of SEQ ID NO: 81. In other aspects of this embodiment, a BMP2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 296-396 of SEQ ID NO: 81. In stillother aspects of this embodiment, a BMP2 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids296-396 of SEQ ID NO: 81. In other aspects of this embodiment, a BMP2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 296-396 of SEQ ID NO: 81.

In other aspects of this embodiment, a BMP2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 296-396 of SEQ ID NO: 81. In other aspects of this embodiment, aBMP2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 296-396 of SEQ ID NO: 81. In yetother aspects of this embodiment, a BMP2 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 296-396 ofSEQ ID NO: 81. In other aspects of this embodiment, a BMP2 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 296-396 of SEQ ID NO: 81. In still other aspects of thisembodiment, a BMP2 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 296-396 of SEQ ID NO: 81.In other aspects of this embodiment, a BMP2 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids296-396 of SEQ ID NO: 81.

In an embodiment, a binding domain comprises a BMP3. In anotherembodiment, a binding domain comprises a BMP3 of SEQ ID NO: 82. Inanother embodiment, a binding domain is derived from a BMP3. In anotherembodiment, a binding domain is derived from a BMP3 of SEQ ID NO: 82. Inan aspect of this embodiment, a binding domain is derived from a BMP3comprising amino acids 370-472 of SEQ ID NO: 82.

In other aspects of this embodiment, a BMP3 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 370-472of SEQ ID NO: 82, at least 75% amino acid identity with amino acids370-472 of SEQ ID NO: 82, at least 80% amino acid identity with aminoacids 370-472 of SEQ ID NO: 82, at least 85% amino acid identity withamino acids 370-472 of SEQ ID NO: 82, at least 90% amino acid identitywith amino acids 370-472 of SEQ ID NO: 82 or at least 95% amino acididentity with amino acids 370-472 of SEQ ID NO: 82. In yet other aspectsof this embodiment, a BMP3 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 370-472 of SEQ ID NO: 82, atmost 75% amino acid identity with amino acids 370-472 of SEQ ID NO: 82,at most 80% amino acid identity with amino acids 370-472 of SEQ ID NO:82, at most 85% amino acid identity with amino acids 370-472 of SEQ IDNO: 82, at most 90% amino acid identity with amino acids 370-472 of SEQID NO: 82 or at most 95% amino acid identity with amino acids 370-472 ofSEQ ID NO: 82.

In other aspects of this embodiment, a BMP3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 370-472 of SEQ ID NO: 82. In other aspects of this embodiment, aBMP3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 370-472 of SEQ ID NO: 82. In yetother aspects of this embodiment, a BMP3 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids370-472 of SEQ ID NO: 82. In other aspects of this embodiment, a BMP3comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 370-472 of SEQ ID NO: 82. In stillother aspects of this embodiment, a BMP3 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids370-472 of SEQ ID NO: 82. In other aspects of this embodiment, a BMP3comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 370-472 of SEQ ID NO: 82.

In other aspects of this embodiment, a BMP3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 370-472 of SEQ ID NO: 82. In other aspects of this embodiment, aBMP3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 370-472 of SEQ ID NO: 82. In yetother aspects of this embodiment, a BMP3 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 370-472 ofSEQ ID NO: 82. In other aspects of this embodiment, a BMP3 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 370-472 of SEQ ID NO: 82. In still other aspects of thisembodiment, a BMP3 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 370-472 of SEQ ID NO: 82.In other aspects of this embodiment, a BMP3 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids370-472 of SEQ ID NO: 82.

In an embodiment, a binding domain comprises a BMP4. In anotherembodiment, a binding domain comprises a BMP4 of SEQ ID NO: 83. Inanother embodiment, a binding domain is derived from a BMP4. In anotherembodiment, a binding domain is derived from a BMP4 of SEQ ID NO: 83. Inan aspect of this embodiment, a binding domain is derived from a BMP4comprising amino acids 309-409 of SEQ ID NO: 83.

In other aspects of this embodiment, a BMP4 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 309-409of SEQ ID NO: 83, at least 75% amino acid identity with amino acids309-409 of SEQ ID NO: 83, at least 80% amino acid identity with aminoacids 309-409 of SEQ ID NO: 83, at least 85% amino acid identity withamino acids 309-409 of SEQ ID NO: 83, at least 90% amino acid identitywith amino acids 309-409 of SEQ ID NO: 83 or at least 95% amino acididentity with amino acids 309-409 of SEQ ID NO: 83. In yet other aspectsof this embodiment, a BMP4 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 309-409 of SEQ ID NO: 83, atmost 75% amino acid identity with amino acids 309-409 of SEQ ID NO: 83,at most 80% amino acid identity with amino acids 309-409 of SEQ ID NO:83, at most 85% amino acid identity with amino acids 309-409 of SEQ IDNO: 83, at most 90% amino acid identity with amino acids 309-409 of SEQID NO: 83 or at most 95% amino acid identity with amino acids 309-409 ofSEQ ID NO: 83.

In other aspects of this embodiment, a BMP4 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 309-409 of SEQ ID NO: 83. In other aspects of this embodiment, aBMP4 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 309-409 of SEQ ID NO: 83. In yetother aspects of this embodiment, a BMP4 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids309-409 of SEQ ID NO: 83. In other aspects of this embodiment, a BMP4comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 309-409 of SEQ ID NO: 83. In stillother aspects of this embodiment, a BMP4 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids309-409 of SEQ ID NO: 83. In other aspects of this embodiment, a BMP4comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 309-409 of SEQ ID NO: 83.

In other aspects of this embodiment, a BMP4 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 309-409 of SEQ ID NO: 83. In other aspects of this embodiment, aBMP4 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 309-409 of SEQ ID NO: 83. In yetother aspects of this embodiment, a BMP4 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 309-409 ofSEQ ID NO: 83. In other aspects of this embodiment, a BMP4 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 309-409 of SEQ ID NO: 83. In still other aspects of thisembodiment, a BMP4 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 309-409 of SEQ ID NO: 83.In other aspects of this embodiment, a BMP4 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids309-409 of SEQ ID NO: 83.

In an embodiment, a binding domain comprises a BMP5. In anotherembodiment, a binding domain comprises a BMP5 of SEQ ID NO: 84. Inanother embodiment, a binding domain is derived from a BMP5. In anotherembodiment, a binding domain is derived from a BMP5 of SEQ ID NO: 84. Inan aspect of this embodiment, a binding domain is derived from a BMP5comprising amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84.

In other aspects of this embodiment, a BMP5 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 353-454or amino acids 323-454 of SEQ ID NO: 84, at least 75% amino acididentity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO:84, at least 80% amino acid identity with amino acids 353-454 or aminoacids 323-454 of SEQ ID NO: 84, at least 85% amino acid identity withamino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84, at least90% amino acid identity with amino acids 353-454 or amino acids 323-454of SEQ ID NO: 84 or at least 95% amino acid identity with amino acids353-454 or amino acids 323-454 of SEQ ID NO: 84. In yet other aspects ofthis embodiment, a BMP5 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 353-454 or amino acids 323-454of SEQ ID NO: 84, at most 75% amino acid identity with amino acids353-454 or amino acids 323-454 of SEQ ID NO: 84, at most 80% amino acididentity with amino acids 353-454 or amino acids 323-454 of SEQ ID NO:84, at most 85% amino acid identity with amino acids 353-454 or aminoacids 323-454 of SEQ ID NO: 84, at most 90% amino acid identity withamino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84 or at most95% amino acid identity with amino acids 353-454 or amino acids 323-454of SEQ ID NO: 84.

In other aspects of this embodiment, a BMP5 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 353-454 or amino acids 323-454 of SEQ ID NO: 84. In other aspectsof this embodiment, a BMP5 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 353-454or amino acids 323-454 of SEQ ID NO: 84. In yet other aspects of thisembodiment, a BMP5 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 353-454 or amino acids323-454 of SEQ ID NO: 84. In other aspects of this embodiment, a BMP5comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 353-454 or amino acids 323-454 of SEQID NO: 84. In still other aspects of this embodiment, a BMP5 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84.In other aspects of this embodiment, a BMP5 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 353-454 or amino acids 323-454 of SEQ ID NO: 84.

In other aspects of this embodiment, a BMP5 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 353-454 or amino acids 323-454 of SEQ ID NO: 84. In other aspectsof this embodiment, a BMP5 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 353-454 oramino acids 323-454 of SEQ ID NO: 84. In yet other aspects of thisembodiment, a BMP5 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 353-454 or amino acids323-454 of SEQ ID NO: 84. In other aspects of this embodiment, a BMP5comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84.In still other aspects of this embodiment, a BMP5 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 353-454 or amino acids 323-454 of SEQ ID NO: 84. In otheraspects of this embodiment, a BMP5 comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 353-454 or aminoacids 323-454 of SEQ ID NO: 84.

In an embodiment, a binding domain comprises a BMP6. In anotherembodiment, a binding domain comprises a BMP6 of SEQ ID NO: 85. Inanother embodiment, a binding domain is derived from a BMP6. In anotherembodiment, a binding domain is derived from a BMP6 of SEQ ID NO: 85. Inan aspect of this embodiment, a binding domain is derived from a BMP6comprising amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85.

In other aspects of this embodiment, a BMP6 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 412-513or amino acids 374-513 of SEQ ID NO: 85, at least 75% amino acididentity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO:85, at least 80% amino acid identity with amino acids 412-513 or aminoacids 374-513 of SEQ ID NO: 85, at least 85% amino acid identity withamino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85, at least90% amino acid identity with amino acids 412-513 or amino acids 374-513of SEQ ID NO: 85 or at least 95% amino acid identity with amino acids412-513 or amino acids 374-513 of SEQ ID NO: 85. In yet other aspects ofthis embodiment, a BMP6 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 412-513 or amino acids 374-513of SEQ ID NO: 85, at most 75% amino acid identity with amino acids412-513 or amino acids 374-513 of SEQ ID NO: 85, at most 80% amino acididentity with amino acids 412-513 or amino acids 374-513 of SEQ ID NO:85, at most 85% amino acid identity with amino acids 412-513 or aminoacids 374-513 of SEQ ID NO: 85, at most 90% amino acid identity withamino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85 or at most95% amino acid identity with amino acids 412-513 or amino acids 374-513of SEQ ID NO: 85.

In other aspects of this embodiment, a BMP6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 412-513 or amino acids 374-513 of SEQ ID NO: 85. In other aspectsof this embodiment, a BMP6 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 412-513or amino acids 374-513 of SEQ ID NO: 85. In yet other aspects of thisembodiment, a BMP6 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 412-513 or amino acids374-513 of SEQ ID NO: 85. In other aspects of this embodiment, a BMP6comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 412-513 or amino acids 374-513 of SEQID NO: 85. In still other aspects of this embodiment, a BMP6 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85.In other aspects of this embodiment, a BMP6 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 412-513 or amino acids 374-513 of SEQ ID NO: 85.

In other aspects of this embodiment, a BMP6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 412-513 or amino acids 374-513 of SEQ ID NO: 85. In other aspectsof this embodiment, a BMP6 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 412-513 oramino acids 374-513 of SEQ ID NO: 85. In yet other aspects of thisembodiment, a BMP6 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 412-513 or amino acids374-513 of SEQ ID NO: 85. In other aspects of this embodiment, a BMP6comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85.In still other aspects of this embodiment, a BMP6 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 412-513 or amino acids 374-513 of SEQ ID NO: 85. In otheraspects of this embodiment, a BMP6 comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 412-513 or aminoacids 374-513 of SEQ ID NO: 85.

In an embodiment, a binding domain comprises a BMP7. In anotherembodiment, a binding domain comprises a BMP7 of SEQ ID NO: 86. Inanother embodiment, a binding domain is derived from a BMP7. In anotherembodiment, a binding domain is derived from a BMP7 of SEQ ID NO: 86. Inan aspect of this embodiment, a binding domain is derived from a BMP7comprising amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86.

In other aspects of this embodiment, a BMP7 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 330-431or amino acids 293-431 of SEQ ID NO: 86, at least 75% amino acididentity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO:86, at least 80% amino acid identity with amino acids 330-431 or aminoacids 293-431 of SEQ ID NO: 86, at least 85% amino acid identity withamino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86, at least90% amino acid identity with amino acids 330-431 or amino acids 293-431of SEQ ID NO: 86 or at least 95% amino acid identity with amino acids330-431 or amino acids 293-431 of SEQ ID NO: 86. In yet other aspects ofthis embodiment, a BMP7 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 330-431 or amino acids 293-431of SEQ ID NO: 86, at most 75% amino acid identity with amino acids330-431 or amino acids 293-431 of SEQ ID NO: 86, at most 80% amino acididentity with amino acids 330-431 or amino acids 293-431 of SEQ ID NO:86, at most 85% amino acid identity with amino acids 330-431 or aminoacids 293-431 of SEQ ID NO: 86, at most 90% amino acid identity withamino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86 or at most95% amino acid identity with amino acids 330-431 or amino acids 293-431of SEQ ID NO: 86.

In other aspects of this embodiment, a BMP7 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 330-431 or amino acids 293-431 of SEQ ID NO: 86. In other aspectsof this embodiment, a BMP7 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 330-431or amino acids 293-431 of SEQ ID NO: 86. In yet other aspects of thisembodiment, a BMP7 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 330-431 or amino acids293-431 of SEQ ID NO: 86. In other aspects of this embodiment, a BMP7comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 330-431 or amino acids 293-431 of SEQID NO: 86. In still other aspects of this embodiment, a BMP7 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86.In other aspects of this embodiment, a BMP7 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 330-431 or amino acids 293-431 of SEQ ID NO: 86.

In other aspects of this embodiment, a BMP7 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 330-431 or amino acids 293-431 of SEQ ID NO: 86. In other aspectsof this embodiment, a BMP7 comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 330-431 oramino acids 293-431 of SEQ ID NO: 86. In yet other aspects of thisembodiment, a BMP7 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 330-431 or amino acids293-431 of SEQ ID NO: 86. In other aspects of this embodiment, a BMP7comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86.In still other aspects of this embodiment, a BMP7 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 330-431 or amino acids 293-431 of SEQ ID NO: 86. In otheraspects of this embodiment, a BMP7 comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 330-431 or aminoacids 293-431 of SEQ ID NO: 86.

In an embodiment, a binding domain comprises a BMP8. In anotherembodiment, a binding domain comprises a BMP8 of SEQ ID NO: 87. Inanother embodiment, a binding domain is derived from a BMP8. In anotherembodiment, a binding domain is derived from a BMP8 of SEQ ID NO: 87. Inan aspect of this embodiment, a binding domain is derived from a BMP8comprising amino acids 301-402 of SEQ ID NO: 87.

In other aspects of this embodiment, a BMP8 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 301-402of SEQ ID NO: 87, at least 75% amino acid identity with amino acids301-402 of SEQ ID NO: 87, at least 80% amino acid identity with aminoacids 301-402 of SEQ ID NO: 87, at least 85% amino acid identity withamino acids 301-402 of SEQ ID NO: 87, at least 90% amino acid identitywith amino acids 301-402 of SEQ ID NO: 87 or at least 95% amino acididentity with amino acids 301-402 of SEQ ID NO: 87. In yet other aspectsof this embodiment, a BMP8 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 301-402 of SEQ ID NO: 87, atmost 75% amino acid identity with amino acids 301-402 of SEQ ID NO: 87,at most 80% amino acid identity with amino acids 301-402 of SEQ ID NO:87, at most 85% amino acid identity with amino acids 301-402 of SEQ IDNO: 87, at most 90% amino acid identity with amino acids 301-402 of SEQID NO: 87 or at most 95% amino acid identity with amino acids 301-402 ofSEQ ID NO: 87.

In other aspects of this embodiment, a BMP8 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 301-402 of SEQ ID NO: 87. In other aspects of this embodiment, aBMP8 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 301-402 of SEQ ID NO: 87. In yetother aspects of this embodiment, a BMP8 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids301-402 of SEQ ID NO: 87. In other aspects of this embodiment, a BMP8comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 301-402 of SEQ ID NO: 87. In stillother aspects of this embodiment, a BMP8 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids301-402 of SEQ ID NO: 87. In other aspects of this embodiment, a BMP8comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 301-402 of SEQ ID NO: 87.

In other aspects of this embodiment, a BMP8 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 301-402 of SEQ ID NO: 87. In other aspects of this embodiment, aBMP8 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 301-402 of SEQ ID NO: 87. In yetother aspects of this embodiment, a BMP8 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 301-402 ofSEQ ID NO: 87. In other aspects of this embodiment, a BMP8 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 301-402 of SEQ ID NO: 87. In still other aspects of thisembodiment, a BMP8 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 301-402 of SEQ ID NO: 87.In other aspects of this embodiment, a BMP8 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids301-402 of SEQ ID NO: 87.

In an embodiment, a binding domain comprises a BMP10. In anotherembodiment, a binding domain comprises a BMP10 of SEQ ID NO: 88. Inanother embodiment, a binding domain is derived from a BMP10. In anotherembodiment, a binding domain is derived from a BMP10 of SEQ ID NO: 88.In an aspect of this embodiment, a binding domain is derived from aBMP10 comprising amino acids 323-424 of SEQ ID NO: 88.

In other aspects of this embodiment, a BMP10 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 323-424of SEQ ID NO: 88, at least 75% amino acid identity with amino acids323-424 of SEQ ID NO: 88, at least 80% amino acid identity with aminoacids 323-424 of SEQ ID NO: 88, at least 85% amino acid identity withamino acids 323-424 of SEQ ID NO: 88, at least 90% amino acid identitywith amino acids 323-424 of SEQ ID NO: 88 or at least 95% amino acididentity with amino acids 323-424 of SEQ ID NO: 88. In yet other aspectsof this embodiment, a BMP10 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 323-424 of SEQ ID NO: 88,at most 75% amino acid identity with amino acids 323-424 of SEQ ID NO:88, at most 80% amino acid identity with amino acids 323-424 of SEQ IDNO: 88, at most 85% amino acid identity with amino acids 323-424 of SEQID NO: 88, at most 90% amino acid identity with amino acids 323-424 ofSEQ ID NO: 88 or at most 95% amino acid identity with amino acids323-424 of SEQ ID NO: 88.

In other aspects of this embodiment, a BMP10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 323-424 of SEQ ID NO: 88. In other aspects of this embodiment, aBMP10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 323-424 of SEQ ID NO: 88. In yetother aspects of this embodiment, a BMP10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 323-424 of SEQ ID NO: 88. In other aspects of this embodiment, aBMP10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 323-424 of SEQ ID NO: 88. In stillother aspects of this embodiment, a BMP10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 323-424 of SEQ ID NO: 88. In other aspects of this embodiment, aBMP10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 323-424 of SEQ ID NO: 88.

In other aspects of this embodiment, a BMP10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 323-424 of SEQ ID NO: 88. In other aspects of this embodiment, aBMP10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 323-424 of SEQ ID NO: 88. In yetother aspects of this embodiment, a BMP10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids323-424 of SEQ ID NO: 88. In other aspects of this embodiment, a BMP10comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 323-424 of SEQ ID NO: 88. In still other aspectsof this embodiment, a BMP10 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 323-424 of SEQID NO: 88. In other aspects of this embodiment, a BMP10 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 323-424 of SEQ ID NO: 88.

Another example of a binding domain disclosed in the presentspecification is, e.g., a GFPs, such as, e.g., GDF1, GDF2, GDF3, GDF5,GDF6, GDF7, GDF8, GDF10, GDF11 or GDF15.

Thus, in an embodiment, a binding domain comprises a GDF1. In anotherembodiment, a binding domain comprises a GDF1 of SEQ ID NO: 89. Inanother embodiment, a binding domain is derived from a GDF1. In anotherembodiment, a binding domain is derived from a GDF1 of SEQ ID NO: 89. Inan aspect of this embodiment, a binding domain is derived from a GDF1comprising amino acids 267-372 of SEQ ID NO: 89.

In other aspects of this embodiment, a GDF1 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 267-372of SEQ ID NO: 89, at least 75% amino acid identity with amino acids267-372 of SEQ ID NO: 89, at least 80% amino acid identity with aminoacids 267-372 of SEQ ID NO: 89, at least 85% amino acid identity withamino acids 267-372 of SEQ ID NO: 89, at least 90% amino acid identitywith amino acids 267-372 of SEQ ID NO: 89 or at least 95% amino acididentity with amino acids 267-372 of SEQ ID NO: 89. In yet other aspectsof this embodiment, a GDF1 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 267-372 of SEQ ID NO: 89, atmost 75% amino acid identity with amino acids 267-372 of SEQ ID NO: 89,at most 80% amino acid identity with amino acids 267-372 of SEQ ID NO:89, at most 85% amino acid identity with amino acids 267-372 of SEQ IDNO: 89, at most 90% amino acid identity with amino acids 267-372 of SEQID NO: 89 or at most 95% amino acid identity with amino acids 267-372 ofSEQ ID NO: 89.

In other aspects of this embodiment, a GDF1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 267-372 of SEQ ID NO: 89. In other aspects of this embodiment, aGDF1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 267-372 of SEQ ID NO: 89. In yetother aspects of this embodiment, a GDF1 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids267-372 of SEQ ID NO: 89. In other aspects of this embodiment, a GDF1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 267-372 of SEQ ID NO: 89. In stillother aspects of this embodiment, a GDF1 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids267-372 of SEQ ID NO: 89. In other aspects of this embodiment, a GDF1comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 267-372 of SEQ ID NO: 89.

In other aspects of this embodiment, a GDF1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 267-372 of SEQ ID NO: 89. In other aspects of this embodiment, aGDF1 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 267-372 of SEQ ID NO: 89. In yetother aspects of this embodiment, a GDF1 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 267-372 ofSEQ ID NO: 89. In other aspects of this embodiment, a GDF1 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 267-372 of SEQ ID NO: 89. In still other aspects of thisembodiment, a GDF1 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 267-372 of SEQ ID NO: 89.In other aspects of this embodiment, a GDF1 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids267-372 of SEQ ID NO: 89.

In an embodiment, a binding domain comprises a GDF2. In anotherembodiment, a binding domain comprises a GDF2 of SEQ ID NO: 90. Inanother embodiment, a binding domain is derived from a GDF2. In anotherembodiment, a binding domain is derived from a GDF2 of SEQ ID NO: 90. Inan aspect of this embodiment, a binding domain is derived from a GDF2comprising amino acids 327-429 of SEQ ID NO: 90.

In other aspects of this embodiment, a GDF2 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 327-429of SEQ ID NO: 90, at least 75% amino acid identity with amino acids327-429 of SEQ ID NO: 90, at least 80% amino acid identity with aminoacids 327-429 of SEQ ID NO: 90, at least 85% amino acid identity withamino acids 327-429 of SEQ ID NO: 90, at least 90% amino acid identitywith amino acids 327-429 of SEQ ID NO: 90 or at least 95% amino acididentity with amino acids 327-429 of SEQ ID NO: 90. In yet other aspectsof this embodiment, a GDF2 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 327-429 of SEQ ID NO: 90, atmost 75% amino acid identity with amino acids 327-429 of SEQ ID NO: 90,at most 80% amino acid identity with amino acids 327-429 of SEQ ID NO:90, at most 85% amino acid identity with amino acids 327-429 of SEQ IDNO: 90, at most 90% amino acid identity with amino acids 327-429 of SEQID NO: 90 or at most 95% amino acid identity with amino acids 327-429 ofSEQ ID NO: 90.

In other aspects of this embodiment, a GDF2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 327-429 of SEQ ID NO: 90. In other aspects of this embodiment, aGDF2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 327-429 of SEQ ID NO: 90. In yetother aspects of this embodiment, a GDF2 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids327-429 of SEQ ID NO: 90. In other aspects of this embodiment, a GDF2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 327-429 of SEQ ID NO: 90. In stillother aspects of this embodiment, a GDF2 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids327-429 of SEQ ID NO: 90. In other aspects of this embodiment, a GDF2comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 327-429 of SEQ ID NO: 90.

In other aspects of this embodiment, a GDF2 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 327-429 of SEQ ID NO: 90. In other aspects of this embodiment, aGDF2 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 327-429 of SEQ ID NO: 90. In yetother aspects of this embodiment, a GDF2 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 327-429 ofSEQ ID NO: 90. In other aspects of this embodiment, a GDF2 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 327-429 of SEQ ID NO: 90. In still other aspects of thisembodiment, a GDF2 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 327-429 of SEQ ID NO: 90.In other aspects of this embodiment, a GDF2 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids327-429 of SEQ ID NO: 90.

In an embodiment, a binding domain comprises a GDF3. In anotherembodiment, a binding domain comprises a GDF3 of SEQ ID NO: 91. Inanother embodiment, a binding domain is derived from a GDF3. In anotherembodiment, a binding domain is derived from a GDF3 of SEQ ID NO: 91. Inan aspect of this embodiment, a binding domain is derived from a GDF3comprising amino acids 264-364 of SEQ ID NO: 91.

In other aspects of this embodiment, a GDF3 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 264-364of SEQ ID NO: 91, at least 75% amino acid identity with amino acids264-364 of SEQ ID NO: 91, at least 80% amino acid identity with aminoacids 264-364 of SEQ ID NO: 91, at least 85% amino acid identity withamino acids 264-364 of SEQ ID NO: 91, at least 90% amino acid identitywith amino acids 264-364 of SEQ ID NO: 91 or at least 95% amino acididentity with amino acids 264-364 of SEQ ID NO: 91. In yet other aspectsof this embodiment, a GDF3 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 264-364 of SEQ ID NO: 91, atmost 75% amino acid identity with amino acids 264-364 of SEQ ID NO: 91,at most 80% amino acid identity with amino acids 264-364 of SEQ ID NO:91, at most 85% amino acid identity with amino acids 264-364 of SEQ IDNO: 91, at most 90% amino acid identity with amino acids 264-364 of SEQID NO: 91 or at most 95% amino acid identity with amino acids 264-364 ofSEQ ID NO: 91.

In other aspects of this embodiment, a GDF3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 264-364 of SEQ ID NO: 91. In other aspects of this embodiment, aGDF3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 264-364 of SEQ ID NO: 91. In yetother aspects of this embodiment, a GDF3 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids264-364 of SEQ ID NO: 91. In other aspects of this embodiment, a GDF3comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 264-364 of SEQ ID NO: 91. In stillother aspects of this embodiment, a GDF3 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids264-364 of SEQ ID NO: 91. In other aspects of this embodiment, a GDF3comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 264-364 of SEQ ID NO: 91.

In other aspects of this embodiment, a GDF3 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 264-364 of SEQ ID NO: 91. In other aspects of this embodiment, aGDF3 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 264-364 of SEQ ID NO: 91. In yetother aspects of this embodiment, a GDF3 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 264-364 ofSEQ ID NO: 91. In other aspects of this embodiment, a GDF3 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 264-364 of SEQ ID NO: 91. In still other aspects of thisembodiment, a GDF3 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 264-364 of SEQ ID NO: 91.In other aspects of this embodiment, a GDF3 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids264-364 of SEQ ID NO: 91.

In an embodiment, a binding domain comprises a GDF5. In anotherembodiment, a binding domain comprises a GDF5 of SEQ ID NO: 92. Inanother embodiment, a binding domain is derived from a GDF5. In anotherembodiment, a binding domain is derived from a GDF5 of SEQ ID NO: 92. Inan aspect of this embodiment, a binding domain is derived from a GDF5comprising amino acids 400-501 of SEQ ID NO: 92.

In other aspects of this embodiment, a GDF5 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 400-501of SEQ ID NO: 92, at least 75% amino acid identity with amino acids400-501 of SEQ ID NO: 92, at least 80% amino acid identity with aminoacids 400-501 of SEQ ID NO: 92, at least 85% amino acid identity withamino acids 400-501 of SEQ ID NO: 92, at least 90% amino acid identitywith amino acids 400-501 of SEQ ID NO: 92 or at least 95% amino acididentity with amino acids 400-501 of SEQ ID NO: 92. In yet other aspectsof this embodiment, a GDF5 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 400-501 of SEQ ID NO: 92, atmost 75% amino acid identity with amino acids 400-501 of SEQ ID NO: 92,at most 80% amino acid identity with amino acids 400-501 of SEQ ID NO:92, at most 85% amino acid identity with amino acids 400-501 of SEQ IDNO: 92, at most 90% amino acid identity with amino acids 400-501 of SEQID NO: 92 or at most 95% amino acid identity with amino acids 400-501 ofSEQ ID NO: 92.

In other aspects of this embodiment, a GDF5 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 400-501 of SEQ ID NO: 92. In other aspects of this embodiment, aGDF5 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 400-501 of SEQ ID NO: 92. In yetother aspects of this embodiment, a GDF5 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids400-501 of SEQ ID NO: 92. In other aspects of this embodiment, a GDF5comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 400-501 of SEQ ID NO: 92. In stillother aspects of this embodiment, a GDF5 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids400-501 of SEQ ID NO: 92. In other aspects of this embodiment, a GDF5comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 400-501 of SEQ ID NO: 92.

In other aspects of this embodiment, a GDF5 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 400-501 of SEQ ID NO: 92. In other aspects of this embodiment, aGDF5 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 400-501 of SEQ ID NO: 92. In yetother aspects of this embodiment, a GDF5 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 400-501 ofSEQ ID NO: 92. In other aspects of this embodiment, a GDF5 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 400-501 of SEQ ID NO: 92. In still other aspects of thisembodiment, a GDF5 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 400-501 of SEQ ID NO: 92.In other aspects of this embodiment, a GDF5 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids400-501 of SEQ ID NO: 92.

In an embodiment, a binding domain comprises a GDF6. In anotherembodiment, a binding domain comprises a GDF6 of SEQ ID NO: 93. Inanother embodiment, a binding domain is derived from a GDF6. In anotherembodiment, a binding domain is derived from a GDF6 of SEQ ID NO: 93. Inan aspect of this embodiment, a binding domain is derived from a GDF6comprising amino acids 354-455 of SEQ ID NO: 93.

In other aspects of this embodiment, a GDF6 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 354-455of SEQ ID NO: 93, at least 75% amino acid identity with amino acids354-455 of SEQ ID NO: 93, at least 80% amino acid identity with aminoacids 354-455 of SEQ ID NO: 93, at least 85% amino acid identity withamino acids 354-455 of SEQ ID NO: 93, at least 90% amino acid identitywith amino acids 354-455 of SEQ ID NO: 93 or at least 95% amino acididentity with amino acids 354-455 of SEQ ID NO: 93. In yet other aspectsof this embodiment, a GDF6 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 354-455 of SEQ ID NO: 93, atmost 75% amino acid identity with amino acids 354-455 of SEQ ID NO: 93,at most 80% amino acid identity with amino acids 354-455 of SEQ ID NO:93, at most 85% amino acid identity with amino acids 354-455 of SEQ IDNO: 93, at most 90% amino acid identity with amino acids 354-455 of SEQID NO: 93 or at most 95% amino acid identity with amino acids 354-455 ofSEQ ID NO: 93.

In other aspects of this embodiment, a GDF6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 354-455 of SEQ ID NO: 93. In other aspects of this embodiment, aGDF6 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 354-455 of SEQ ID NO: 93. In yetother aspects of this embodiment, a GDF6 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids354-455 of SEQ ID NO: 93. In other aspects of this embodiment, a GDF6comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 354-455 of SEQ ID NO: 93. In stillother aspects of this embodiment, a GDF6 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids354-455 of SEQ ID NO: 93. In other aspects of this embodiment, a GDF6comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 354-455 of SEQ ID NO: 93.

In other aspects of this embodiment, a GDF6 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 354-455 of SEQ ID NO: 93. In other aspects of this embodiment, aGDF6 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 354-455 of SEQ ID NO: 93. In yetother aspects of this embodiment, a GDF6 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 354-455 ofSEQ ID NO: 93. In other aspects of this embodiment, a GDF6 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 354-455 of SEQ ID NO: 93. In still other aspects of thisembodiment, a GDF6 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 354-455 of SEQ ID NO: 93.In other aspects of this embodiment, a GDF6 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids354-455 of SEQ ID NO: 93.

In an embodiment, a binding domain comprises a GDF7. In anotherembodiment, a binding domain comprises a GDF7 of SEQ ID NO: 94. Inanother embodiment, a binding domain is derived from a GDF7. In anotherembodiment, a binding domain is derived from a GDF7 of SEQ ID NO: 94. Inan aspect of this embodiment, a binding domain is derived from a GDF7comprising amino acids 352-450 of SEQ ID NO: 94.

In other aspects of this embodiment, a GDF7 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 352-450of SEQ ID NO: 94, at least 75% amino acid identity with amino acids352-450 of SEQ ID NO: 94, at least 80% amino acid identity with aminoacids 352-450 of SEQ ID NO: 94, at least 85% amino acid identity withamino acids 352-450 of SEQ ID NO: 94, at least 90% amino acid identitywith amino acids 352-450 of SEQ ID NO: 94 or at least 95% amino acididentity with amino acids 352-450 of SEQ ID NO: 94. In yet other aspectsof this embodiment, a GDF7 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 352-450 of SEQ ID NO: 94, atmost 75% amino acid identity with amino acids 352-450 of SEQ ID NO: 94,at most 80% amino acid identity with amino acids 352-450 of SEQ ID NO:94, at most 85% amino acid identity with amino acids 352-450 of SEQ IDNO: 94, at most 90% amino acid identity with amino acids 352-450 of SEQID NO: 94 or at most 95% amino acid identity with amino acids 352-450 ofSEQ ID NO: 94.

In other aspects of this embodiment, a GDF7 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 352-450 of SEQ ID NO: 94. In other aspects of this embodiment, aGDF7 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 352-450 of SEQ ID NO: 94. In yetother aspects of this embodiment, a GDF7 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids352-450 of SEQ ID NO: 94. In other aspects of this embodiment, a GDF7comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 352-450 of SEQ ID NO: 94. In stillother aspects of this embodiment, a GDF7 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids352-450 of SEQ ID NO: 94. In other aspects of this embodiment, a GDF7comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 352-450 of SEQ ID NO: 94.

In other aspects of this embodiment, a GDF7 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 352-450 of SEQ ID NO: 94. In other aspects of this embodiment, aGDF7 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 352-450 of SEQ ID NO: 94. In yetother aspects of this embodiment, a GDF7 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 352-450 ofSEQ ID NO: 94. In other aspects of this embodiment, a GDF7 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 352-450 of SEQ ID NO: 94. In still other aspects of thisembodiment, a GDF7 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 352-450 of SEQ ID NO: 94.In other aspects of this embodiment, a GDF7 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids352-450 of SEQ ID NO: 94.

In an embodiment, a binding domain comprises a GDF8. In anotherembodiment, a binding domain comprises a GDF8 of SEQ ID NO: 95. Inanother embodiment, a binding domain is derived from a GDF8. In anotherembodiment, a binding domain is derived from a GDF8 of SEQ ID NO: 95. Inan aspect of this embodiment, a binding domain is derived from a GDF8comprising amino acids 281-375 of SEQ ID NO: 95.

In other aspects of this embodiment, a GDF8 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 281-375of SEQ ID NO: 95, at least 75% amino acid identity with amino acids281-375 of SEQ ID NO: 95, at least 80% amino acid identity with aminoacids 281-375 of SEQ ID NO: 95, at least 85% amino acid identity withamino acids 281-375 of SEQ ID NO: 95, at least 90% amino acid identitywith amino acids 281-375 of SEQ ID NO: 95 or at least 95% amino acididentity with amino acids 281-375 of SEQ ID NO: 95. In yet other aspectsof this embodiment, a GDF8 comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 281-375 of SEQ ID NO: 95, atmost 75% amino acid identity with amino acids 281-375 of SEQ ID NO: 95,at most 80% amino acid identity with amino acids 281-375 of SEQ ID NO:95, at most 85% amino acid identity with amino acids 281-375 of SEQ IDNO: 95, at most 90% amino acid identity with amino acids 281-375 of SEQID NO: 95 or at most 95% amino acid identity with amino acids 281-375 ofSEQ ID NO: 95.

In other aspects of this embodiment, a GDF8 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 281-375 of SEQ ID NO: 95. In other aspects of this embodiment, aGDF8 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 281-375 of SEQ ID NO: 95. In yetother aspects of this embodiment, a GDF8 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids281-375 of SEQ ID NO: 95. In other aspects of this embodiment, a GDF8comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 281-375 of SEQ ID NO: 95. In stillother aspects of this embodiment, a GDF8 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids281-375 of SEQ ID NO: 95. In other aspects of this embodiment, a GDF8comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 281-375 of SEQ ID NO: 95.

In other aspects of this embodiment, a GDF8 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 281-375 of SEQ ID NO: 95. In other aspects of this embodiment, aGDF8 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 281-375 of SEQ ID NO: 95. In yetother aspects of this embodiment, a GDF8 comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 281-375 ofSEQ ID NO: 95. In other aspects of this embodiment, a GDF8 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 281-375 of SEQ ID NO: 95. In still other aspects of thisembodiment, a GDF8 comprises a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid additions relative to amino acids 281-375 of SEQ ID NO: 95.In other aspects of this embodiment, a GDF8 comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids281-375 of SEQ ID NO: 95.

In an embodiment, a binding domain comprises a GDF10. In anotherembodiment, a binding domain comprises a GDF19 of SEQ ID NO: 96. Inanother embodiment, a binding domain is derived from a GDF10. In anotherembodiment, a binding domain is derived from a GDF10 of SEQ ID NO: 96.In an aspect of this embodiment, a binding domain is derived from aGDF10 comprising amino acids 376-478 of SEQ ID NO: 96.

In other aspects of this embodiment, a GDF10 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 376-478of SEQ ID NO: 96, at least 75% amino acid identity with amino acids376-478 of SEQ ID NO: 96, at least 80% amino acid identity with aminoacids 376-478 of SEQ ID NO: 96, at least 85% amino acid identity withamino acids 376-478 of SEQ ID NO: 96, at least 90% amino acid identitywith amino acids 376-478 of SEQ ID NO: 96 or at least 95% amino acididentity with amino acids 376-478 of SEQ ID NO: 96. In yet other aspectsof this embodiment, a GDF10 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 376-478 of SEQ ID NO: 96,at most 75% amino acid identity with amino acids 376-478 of SEQ ID NO:96, at most 80% amino acid identity with amino acids 376-478 of SEQ IDNO: 96, at most 85% amino acid identity with amino acids 376-478 of SEQID NO: 96, at most 90% amino acid identity with amino acids 376-478 ofSEQ ID NO: 96 or at most 95% amino acid identity with amino acids376-478 of SEQ ID NO: 96.

In other aspects of this embodiment, a GDF10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 376-478 of SEQ ID NO: 96. In other aspects of this embodiment, aGDF10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 376-478 of SEQ ID NO: 96. In yetother aspects of this embodiment, a GDF10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 376-478 of SEQ ID NO: 96. In other aspects of this embodiment, aGDF10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 376-478 of SEQ ID NO: 96. In stillother aspects of this embodiment, a GDF10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 376-478 of SEQ ID NO: 96. In other aspects of this embodiment, aGDF10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 376-478 of SEQ ID NO: 96.

In other aspects of this embodiment, a GDF10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 376-478 of SEQ ID NO: 96. In other aspects of this embodiment, aGDF10 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 376-478 of SEQ ID NO: 96. In yetother aspects of this embodiment, a GDF10 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids376-478 of SEQ ID NO: 96. In other aspects of this embodiment, a GDF10comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 376-478 of SEQ ID NO: 96. In still other aspectsof this embodiment, a GDF10 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 376-478 of SEQID NO: 96. In other aspects of this embodiment, a GDF10 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 376-478 of SEQ ID NO: 96.

In an embodiment, a binding domain comprises a GDF11. In anotherembodiment, a binding domain comprises a GDF11 of SEQ ID NO: 97. Inanother embodiment, a binding domain is derived from a GDF11. In anotherembodiment, a binding domain is derived from a GDF11 of SEQ ID NO: 97.In an aspect of this embodiment, a binding domain is derived from aGDF11 comprising amino acids 313-407 of SEQ ID NO: 97.

In other aspects of this embodiment, a GDF11 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 313-407of SEQ ID NO: 97, at least 75% amino acid identity with amino acids313-407 of SEQ ID NO: 97, at least 80% amino acid identity with aminoacids 313-407 of SEQ ID NO: 97, at least 85% amino acid identity withamino acids 313-407 of SEQ ID NO: 97, at least 90% amino acid identitywith amino acids 313-407 of SEQ ID NO: 97 or at least 95% amino acididentity with amino acids 313-407 of SEQ ID NO: 97. In yet other aspectsof this embodiment, a GDF11 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 313-407 of SEQ ID NO: 97,at most 75% amino acid identity with amino acids 313-407 of SEQ ID NO:97, at most 80% amino acid identity with amino acids 313-407 of SEQ IDNO: 97, at most 85% amino acid identity with amino acids 313-407 of SEQID NO: 97, at most 90% amino acid identity with amino acids 313-407 ofSEQ ID NO: 97 or at most 95% amino acid identity with amino acids313-407 of SEQ ID NO: 97.

In other aspects of this embodiment, a GDF11 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 313-407 of SEQ ID NO: 97. In other aspects of this embodiment, aGDF11 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 313-407 of SEQ ID NO: 97. In yetother aspects of this embodiment, a GDF11 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 313-407 of SEQ ID NO: 97. In other aspects of this embodiment, aGDF11 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 313-407 of SEQ ID NO: 97. In stillother aspects of this embodiment, a GDF11 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 313-407 of SEQ ID NO: 97. In other aspects of this embodiment, aGDF11 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 313-407 of SEQ ID NO: 97.

In other aspects of this embodiment, a GDF11 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 313-407 of SEQ ID NO: 97. In other aspects of this embodiment, aGDF11 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 313-407 of SEQ ID NO: 97. In yetother aspects of this embodiment, a GDF11 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids313-407 of SEQ ID NO: 97. In other aspects of this embodiment, a GDF11comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 313-407 of SEQ ID NO: 97. In still other aspectsof this embodiment, a GDF11 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 313-407 of SEQID NO: 97. In other aspects of this embodiment, a GDF11 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 313-407 of SEQ ID NO: 97.

In an embodiment, a binding domain comprises a GDF15. In anotherembodiment, a binding domain comprises a GDF15 of SEQ ID NO: 98. Inanother embodiment, a binding domain is derived from a GDF15. In anotherembodiment, a binding domain is derived from a GDF15 of SEQ ID NO: 98.In an aspect of this embodiment, a binding domain is derived from aGDF15 comprising amino acids 211-308 of SEQ ID NO: 98.

In other aspects of this embodiment, a GDF15 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with amino acids 211-308of SEQ ID NO: 98, at least 75% amino acid identity with amino acids211-308 of SEQ ID NO: 98, at least 80% amino acid identity with aminoacids 211-308 of SEQ ID NO: 98, at least 85% amino acid identity withamino acids 211-308 of SEQ ID NO: 98, at least 90% amino acid identitywith amino acids 211-308 of SEQ ID NO: 98 or at least 95% amino acididentity with amino acids 211-308 of SEQ ID NO: 98. In yet other aspectsof this embodiment, a GDF15 comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 211-308 of SEQ ID NO: 98,at most 75% amino acid identity with amino acids 211-308 of SEQ ID NO:98, at most 80% amino acid identity with amino acids 211-308 of SEQ IDNO: 98, at most 85% amino acid identity with amino acids 211-308 of SEQID NO: 98, at most 90% amino acid identity with amino acids 211-308 ofSEQ ID NO: 98 or at most 95% amino acid identity with amino acids211-308 of SEQ ID NO: 98.

In other aspects of this embodiment, a GDF15 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 211-308 of SEQ ID NO: 98. In other aspects of this embodiment, aGDF15 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 211-308 of SEQ ID NO: 98. In yetother aspects of this embodiment, a GDF15 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 211-308 of SEQ ID NO: 98. In other aspects of this embodiment, aGDF15 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 211-308 of SEQ ID NO: 98. In stillother aspects of this embodiment, a GDF15 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 211-308 of SEQ ID NO: 98. In other aspects of this embodiment, aGDF15 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 211-308 of SEQ ID NO: 98.

In other aspects of this embodiment, a GDF15 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 211-308 of SEQ ID NO: 98. In other aspects of this embodiment, aGDF15 comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 211-308 of SEQ ID NO: 98. In yetother aspects of this embodiment, a GDF15 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids211-308 of SEQ ID NO: 98. In other aspects of this embodiment, a GDF15comprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 211-308 of SEQ ID NO: 98. In still other aspectsof this embodiment, a GDF15 comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 211-308 of SEQID NO: 98. In other aspects of this embodiment, a GDF15 comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid additions relative toamino acids 211-308 of SEQ ID NO: 98.

Another example of a binding domain disclosed in the presentspecification is, e.g., an activin A, an activin B, an activin C, anactivin E or an inhibin A.

Thus. in an embodiment, a binding domain comprises an Activin A. Inanother embodiment, a binding domain comprises an Activin A of SEQ IDNO: 99. In another embodiment, a binding domain is derived from anActivin A. In another embodiment, a binding domain is derived from anActivin A of SEQ ID NO: 99. In an aspect of this embodiment, a bindingdomain is derived from an Activin A comprising amino acids 321-426 ofSEQ ID NO: 99.

In other aspects of this embodiment, an Activin A comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 321-426 of SEQ ID NO: 99, at least 75% amino acid identity withamino acids 321-426 of SEQ ID NO: 99, at least 80% amino acid identitywith amino acids 321-426 of SEQ ID NO: 99, at least 85% amino acididentity with amino acids 321-426 of SEQ ID NO: 99, at least 90% aminoacid identity with amino acids 321-426 of SEQ ID NO: 99 or at least 95%amino acid identity with amino acids 321-426 of SEQ ID NO: 99. In yetother aspects of this embodiment, an Activin A comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 321-426of SEQ ID NO: 99, at most 75% amino acid identity with amino acids321-426 of SEQ ID NO: 99, at most 80% amino acid identity with aminoacids 321-426 of SEQ ID NO: 99, at most 85% amino acid identity withamino acids 321-426 of SEQ ID NO: 99, at most 90% amino acid identitywith amino acids 321-426 of SEQ ID NO: 99 or at most 95% amino acididentity with amino acids 321-426 of SEQ ID NO: 99.

In other aspects of this embodiment, an Activin A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 321-426 of SEQ ID NO: 99. In other aspects ofthis embodiment, an Activin A comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 321-426of SEQ ID NO: 99. In yet other aspects of this embodiment, an Activin Acomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 321-426 of SEQ ID NO: 99. In otheraspects of this embodiment, an Activin A comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids321-426 of SEQ ID NO: 99. In still other aspects of this embodiment, anActivin A comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 321-426 of SEQ ID NO: 99. In otheraspects of this embodiment, an Activin A comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids321-426 of SEQ ID NO: 99.

In other aspects of this embodiment, an Activin A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 321-426 of SEQ ID NO: 99. In other aspects ofthis embodiment, an Activin A comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 321-426 ofSEQ ID NO: 99. In yet other aspects of this embodiment, an Activin Acomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 321-426 of SEQ ID NO: 99. In other aspects ofthis embodiment, an Activin A comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 321-426 of SEQID NO: 99. In still other aspects of this embodiment, an Activin Acomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 321-426 of SEQ ID NO: 99. In other aspects ofthis embodiment, an Activin A comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 321-426 of SEQID NO: 99.

In an embodiment, a binding domain comprises an Activin B. In anotherembodiment, a binding domain comprises an Activin B of SEQ ID NO: 100.In another embodiment, a binding domain is derived from an Activin B. Inanother embodiment, a binding domain is derived from an Activin B of SEQID NO: 100. In an aspect of this embodiment, a binding domain is derivedfrom an Activin B comprising amino acids 303-406 of SEQ ID NO: 100.

In other aspects of this embodiment, an Activin B comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 303-406 of SEQ ID NO: 100, at least 75% amino acid identity withamino acids 303-406 of SEQ ID NO: 100, at least 80% amino acid identitywith amino acids 303-406 of SEQ ID NO: 100, at least 85% amino acididentity with amino acids 303-406 of SEQ ID NO: 100, at least 90% aminoacid identity with amino acids 303-406 of SEQ ID NO: 100 or at least 95%amino acid identity with amino acids 303-406 of SEQ ID NO: 100. In yetother aspects of this embodiment, an Activin B comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 303-406of SEQ ID NO: 100, at most 75% amino acid identity with amino acids303-406 of SEQ ID NO: 100, at most 80% amino acid identity with aminoacids 303-406 of SEQ ID NO: 100, at most 85% amino acid identity withamino acids 303-406 of SEQ ID NO: 100, at most 90% amino acid identitywith amino acids 303-406 of SEQ ID NO: 100 or at most 95% amino acididentity with amino acids 303-406 of SEQ ID NO: 100.

In other aspects of this embodiment, an Activin B comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 303-406 of SEQ ID NO: 100. In other aspects ofthis embodiment, an Activin B comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 303-406of SEQ ID NO: 100. In yet other aspects of this embodiment, an Activin Bcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 303-406 of SEQ ID NO: 100. In otheraspects of this embodiment, an Activin B comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids303-406 of SEQ ID NO: 100. In still other aspects of this embodiment, anActivin B comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 303-406 of SEQ ID NO: 100. In otheraspects of this embodiment, an Activin B comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids303-406 of SEQ ID NO: 100.

In other aspects of this embodiment, an Activin B comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 303-406 of SEQ ID NO: 100. In other aspects ofthis embodiment, an Activin B comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 303-406 ofSEQ ID NO: 100. In yet other aspects of this embodiment, an Activin Bcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 303-406 of SEQ ID NO: 100. In other aspects ofthis embodiment, an Activin B comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 303-406 of SEQID NO: 100. In still other aspects of this embodiment, an Activin Bcomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 303-406 of SEQ ID NO: 100. In other aspects ofthis embodiment, an Activin B comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 303-406 of SEQID NO: 100.

In an embodiment, a binding domain comprises an Activin C. In anotherembodiment, a binding domain comprises an Activin C of SEQ ID NO: 101.In another embodiment, a binding domain is derived from an Activin C. Inanother embodiment, a binding domain is derived from an Activin C of SEQID NO: 101. In an aspect of this embodiment, a binding domain is derivedfrom an Activin C comprising amino acids 247-352 or amino acids 237-352of SEQ ID NO: 101.

In other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 247-352 or amino acids 237-352 of SEQ ID NO: 101, at least 75%amino acid identity with amino acids 247-352 or amino acids 237-352 ofSEQ ID NO: 101, at least 80% amino acid identity with amino acids247-352 or amino acids 237-352 of SEQ ID NO: 101, at least 85% aminoacid identity with amino acids 247-352 or amino acids 237-352 of SEQ IDNO: 101, at least 90% amino acid identity with amino acids 247-352 oramino acids 237-352 of SEQ ID NO: 101 or at least 95% amino acididentity with amino acids 247-352 or amino acids 237-352 of SEQ ID NO:101. In yet other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 247-352 or amino acids 237-352 of SEQ ID NO: 101, at most 75%amino acid identity with amino acids 247-352 or amino acids 237-352 ofSEQ ID NO: 101, at most 80% amino acid identity with amino acids 247-352or amino acids 237-352 of SEQ ID NO: 101, at most 85% amino acididentity with amino acids 247-352 or amino acids 237-352 of SEQ ID NO:101, at most 90% amino acid identity with amino acids 247-352 or aminoacids 237-352 of SEQ ID NO: 101 or at most 95% amino acid identity withamino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101.

In other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO:101. In other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO:101. In yet other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO:101. In other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO:101. In still other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO:101. In other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO:101.

In other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO:101. In other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 247-352 or amino acids 237-352 of SEQ ID NO:101. In yet other aspects of this embodiment, an Activin C comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 247-352 or amino acids 237-352 of SEQ ID NO: 101. In otheraspects of this embodiment, an Activin C comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 247-352 oramino acids 237-352 of SEQ ID NO: 101. In still other aspects of thisembodiment, an Activin C comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 247-352 or aminoacids 237-352 of SEQ ID NO: 101. In other aspects of this embodiment, anActivin C comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 247-352 or amino acids 237-352 ofSEQ ID NO: 101.

In an embodiment, a binding domain comprises an Activin D. In anotherembodiment, a binding domain comprises an Activin D of SEQ ID NO: 102.In another embodiment, a binding domain is derived from an Activin E. Inanother embodiment, a binding domain is derived from an Activin E of SEQID NO: 102. In an aspect of this embodiment, a binding domain is derivedfrom an Activin E comprising amino acids 247-350 of SEQ ID NO: 102.

In other aspects of this embodiment, an Activin E comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 247-350 of SEQ ID NO: 102, at least 75% amino acid identity withamino acids 247-350 of SEQ ID NO: 102, at least 80% amino acid identitywith amino acids 247-350 of SEQ ID NO: 102, at least 85% amino acididentity with amino acids 247-350 of SEQ ID NO: 102, at least 90% aminoacid identity with amino acids 247-350 of SEQ ID NO: 102 or at least 95%amino acid identity with amino acids 247-350 of SEQ ID NO: 102. In yetother aspects of this embodiment, an Activin E comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 247-350of SEQ ID NO: 102, at most 75% amino acid identity with amino acids247-350 of SEQ ID NO: 102, at most 80% amino acid identity with aminoacids 247-350 of SEQ ID NO: 102, at most 85% amino acid identity withamino acids 247-350 of SEQ ID NO: 102, at most 90% amino acid identitywith amino acids 247-350 of SEQ ID NO: 102 or at most 95% amino acididentity with amino acids 247-350 of SEQ ID NO: 102.

In other aspects of this embodiment, an Activin E comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 247-350 of SEQ ID NO: 102. In other aspects ofthis embodiment, an Activin E comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 247-350of SEQ ID NO: 102. In yet other aspects of this embodiment, an Activin Ecomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 247-350 of SEQ ID NO: 102. In otheraspects of this embodiment, an Activin E comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid deletions relative to amino acids247-350 of SEQ ID NO: 102. In still other aspects of this embodiment, anActivin E comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 247-350 of SEQ ID NO: 102. In otheraspects of this embodiment, an Activin E comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids247-350 of SEQ ID NO: 102.

In other aspects of this embodiment, an Activin E comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 247-350 of SEQ ID NO: 102. In other aspects ofthis embodiment, an Activin E comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 247-350 ofSEQ ID NO: 102. In yet other aspects of this embodiment, an Activin Ecomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 247-350 of SEQ ID NO: 102. In other aspects ofthis embodiment, an Activin E comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 247-350 of SEQID NO: 102. In still other aspects of this embodiment, an Activin Ecomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 247-350 of SEQ ID NO: 102. In other aspects ofthis embodiment, an Activin E comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 247-350 of SEQID NO: 102.

In an embodiment, a binding domain comprises an Inhibin A. In anotherembodiment, a binding domain comprises an Inhibin A of SEQ ID NO: 103.In another embodiment, a binding domain is derived from an Inhibin A. Inanother embodiment, a binding domain is derived from an Inhibin A of SEQID NO: 103. In an aspect of this embodiment, a binding domain is derivedfrom an Inhibin A comprising amino acids 262-366 or amino acids 233-366of SEQ ID NO: 103.

In other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 262-366 or amino acids 233-366 of SEQ ID NO: 103, at least 75%amino acid identity with amino acids 262-366 or amino acids 233-366 ofSEQ ID NO: 103, at least 80% amino acid identity with amino acids262-366 or amino acids 233-366 of SEQ ID NO: 103, at least 85% aminoacid identity with amino acids 262-366 or amino acids 233-366 of SEQ IDNO: 103, at least 90% amino acid identity with amino acids 262-366 oramino acids 233-366 of SEQ ID NO: 103 or at least 95% amino acididentity with amino acids 262-366 or amino acids 233-366 of SEQ ID NO:103. In yet other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 262-366 or amino acids 233-366 of SEQ ID NO: 103, at most 75%amino acid identity with amino acids 262-366 or amino acids 233-366 ofSEQ ID NO: 103, at most 80% amino acid identity with amino acids 262-366or amino acids 233-366 of SEQ ID NO: 103, at most 85% amino acididentity with amino acids 262-366 or amino acids 233-366 of SEQ ID NO:103, at most 90% amino acid identity with amino acids 262-366 or aminoacids 233-366 of SEQ ID NO: 103 or at most 95% amino acid identity withamino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103.

In other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO:103. In other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid substitutionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO:103. In yet other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO:103. In other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO:103. In still other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO:103. In other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid additionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO:103.

In other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO:103. In other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 262-366 or amino acids 233-366 of SEQ ID NO:103. In yet other aspects of this embodiment, an Inhibin A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 262-366 or amino acids 233-366 of SEQ ID NO: 103. In otheraspects of this embodiment, an Inhibin A comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 262-366 oramino acids 233-366 of SEQ ID NO: 103. In still other aspects of thisembodiment, an Inhibin A comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 262-366 or aminoacids 233-366 of SEQ ID NO: 103. In other aspects of this embodiment, anInhibin A comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 262-366 or amino acids 233-366 ofSEQ ID NO: 103.

Another example of a binding domain includes, without limitation, aopioid peptide, such as, e.g., an enkephalin, an endomorphin, anendorphin, a dynorphin, a nociceptin or a hemorphin. Thus, in anembodiment, a binding domain comprises an opioid peptide. In anotherembodiment, a binding domain is derived from an opioid peptide.

In another embodiment, an opioid binding domain comprises an enkephalinpeptide. In aspects of this embodiment, an opioid binding domain isderived from an enkephalin peptide. In other aspects of this embodiment,an enkephalin binding domain is derived from a Leu-enkephalin, aMet-enkephalin, a Met-enkephalin MRGL or a Met-enkephalin MRF. In otheraspects of this embodiment, an enkephalin binding domain comprises SEQID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107.

In other aspects of this embodiment, an enkephalin binding domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107,at least 75% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105,SEQ ID NO: 106 or SEQ ID NO: 107, at least 80% amino acid identity withSEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107, atleast 85% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQID NO: 106 or SEQ ID NO: 107, at least 90% amino acid identity with SEQID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107 or at least95% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO:106 or SEQ ID NO: 107. In yet other aspects of this embodiment, anenkephalin binding domain comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO:106 or SEQ ID NO: 107, at most 75% amino acid identity with SEQ ID NO:104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107, at most 80% aminoacid identity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQID NO: 107, at most 85% amino acid identity with SEQ ID NO: 104, SEQ IDNO: 105, SEQ ID NO: 106 or SEQ ID NO: 107, at most 90% amino acididentity with SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ IDNO: 107 or at most 95% amino acid identity with SEQ ID NO: 104, SEQ IDNO: 105, SEQ ID NO: 106 or SEQ ID NO: 107.

In other aspects of this embodiment, an enkephalin binding domaincomprises a polypeptide having, e.g., at least one, two or threenon-contiguous amino acid substitutions relative to SEQ ID NO: 104, SEQID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. In other aspects of thisembodiment, an enkephalin binding domain comprises a polypeptide having,e.g., at most one, two or three non-contiguous amino acid substitutionsrelative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO:107. In yet other aspects of this embodiment, an enkephalin bindingdomain comprises a polypeptide having, e.g., at least one, two or threenon-contiguous amino acid deletions relative to SEQ ID NO: 104, SEQ IDNO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. In yet other aspects of thisembodiment, an enkephalin binding domain comprises a polypeptide having,e.g., at most one, two or three non-contiguous amino acid deletionsrelative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO:107. In still other aspects of this embodiment, an enkephalin bindingdomain comprises a polypeptide having, e.g., at least one, two or threenon-contiguous amino acid additions relative to SEQ ID NO: 104, SEQ IDNO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. In yet other aspects of thisembodiment, an enkephalin binding domain comprises a polypeptide having,e.g., at most one, two or three non-contiguous amino acid additionsrelative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO:107.

In other aspects of this embodiment, an enkephalin binding domaincomprises a polypeptide having, e.g., at least one, two or threecontiguous amino acid substitutions relative to SEQ ID NO: 104, SEQ IDNO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. In other aspects of thisembodiment, an enkephalin binding domain comprises a polypeptide having,e.g., at most one, two or three contiguous amino acid substitutionsrelative to SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO:107. In yet other aspects of this embodiment, an enkephalin bindingdomain comprises a polypeptide having, e.g., at least one, two or threecontiguous amino acid deletions relative to SEQ ID NO: 104, SEQ ID NO:105, SEQ ID NO: 106 or SEQ ID NO: 107. In yet other aspects of thisembodiment, an enkephalin binding domain comprises a polypeptide having,e.g., at most one, two or three contiguous amino acid deletions relativeto SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107. Instill other aspects of this embodiment, an enkephalin binding domaincomprises a polypeptide having, e.g., at least one, two or threecontiguous amino acid additions relative to SEQ ID NO: 104, SEQ ID NO:105, SEQ ID NO: 106 or SEQ ID NO: 107. In yet other aspects of thisembodiment, an enkephalin binding domain comprises a polypeptide having,e.g., at most one, two or three contiguous amino acid additions relativeto SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107.

In another embodiment, an opioid binding domain comprises a bovineadrenomedullary-22 (BAM22) peptide. In aspects of this embodiment, anopioid binding domain comprising a BAM22 peptide is derived from a BAM22peptide (1-12), a BAM22 peptide (6-22), a BAM22 peptide (8-22) or aBAM22 peptide (1-22). In other aspects of this embodiment, a BAM22binding domain comprises amino acids 1-12, amino acids 6-22, amino acids8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112 or aminoacids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 ofSEQ ID NO: 113.

In other aspects of this embodiment, a BAM22 binding domain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 ofSEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 oramino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22,amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ IDNO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or aminoacids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22,amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113, at least 75%amino acid identity with amino acids 1-12, amino acids 6-22, amino acids8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or aminoacids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 ofSEQ ID NO: 113, at least 80% amino acid identity with amino acids 1-12,amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO:108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids8-22 or amino acids 1-22 of SEQ ID NO: 113, at least 85% amino acididentity with amino acids 1-12, amino acids 6-22, amino acids 8-22 oramino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22,amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ IDNO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or aminoacids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, aminoacids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12,amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO:113, at least 90% amino acid identity with amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; aminoacids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 ofSEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 oramino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22,amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ IDNO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 oramino acids 1-22 of SEQ ID NO: 113 or at least 95% amino acid identitywith amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113.

In yet other aspects of this embodiment, a BAM22 binding domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113, at most75% amino acid identity with amino acids 1-12, amino acids 6-22, aminoacids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12,amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO:109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; oramino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 113, at most 80% amino acid identity with amino acids1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ IDNO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or aminoacids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, aminoacids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12,amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO:111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, aminoacids 8-22 or amino acids 1-22 of SEQ ID NO: 113, at most 85% amino acididentity with amino acids 1-12, amino acids 6-22, amino acids 8-22 oramino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22,amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ IDNO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or aminoacids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, aminoacids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12,amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO:113, at most 90% amino acid identity with amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108; aminoacids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 ofSEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22 oramino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22,amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ IDNO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22 oramino acids 1-22 of SEQ ID NO: 113 or at most 95% amino acid identitywith amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113.

In other aspects of this embodiment, a BAM22 binding domain comprises apolypeptide having, e.g., at least one, two, three, four or fivenon-contiguous amino acid substitutions relative to amino acids 1-12,amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO:108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids8-22 or amino acids 1-22 of SEQ ID NO: 113. In other aspects of thisembodiment, a BAM22 binding domain comprises a polypeptide having, e.g.,at most one, two, three, four or five non-contiguous amino acidsubstitutions relative to amino acids 1-12, amino acids 6-22, aminoacids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12,amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO:109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; oramino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 113. In yet other aspects of this embodiment, a BAM22binding domain comprises a polypeptide having, e.g., at least one, two,three, four or five non-contiguous amino acid deletions relative toamino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; aminoacids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 ofSEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 oramino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113. In yetother aspects of this embodiment, a BAM22 binding domain comprises apolypeptide having, e.g., at most one, two, three, four or fivenon-contiguous amino acid deletions relative to amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; aminoacids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 ofSEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 113. In still other aspects of thisembodiment, a BAM22 binding domain comprises a polypeptide having, e.g.,at least one, two, three, four or five non-contiguous amino acidadditions relative to amino acids 1-12, amino acids 6-22, amino acids8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or aminoacids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 ofSEQ ID NO: 113. In yet other aspects of this embodiment, a BAM22 bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four or five non-contiguous amino acid additions relative to amino acids1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ IDNO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or aminoacids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, aminoacids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12,amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO:111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, aminoacids 8-22 or amino acids 1-22 of SEQ ID NO: 113.

In other aspects of this embodiment, a BAM22 binding domain comprises apolypeptide having, e.g., at least one, two, three, four or fivecontiguous amino acid substitutions relative to amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; aminoacids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 ofSEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 113. In other aspects of thisembodiment, a BAM22 binding domain comprises a polypeptide having, e.g.,at most one, two, three, four or five contiguous amino acidsubstitutions relative to amino acids 1-12, amino acids 6-22, aminoacids 8-22 or amino acids 1-22 of SEQ ID NO: 108; amino acids 1-12,amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO:109; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids 6-22, amino acids8-22 or amino acids 1-22 of SEQ ID NO: 111; amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 112; oramino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 113. In yet other aspects of this embodiment, a BAM22binding domain comprises a polypeptide having, e.g., at least one, two,three, four or five contiguous amino acid deletions relative to aminoacids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 ofSEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 oramino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22,amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ IDNO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or aminoacids 1-22 of SEQ ID NO: 112; or amino acids 1-12, amino acids 6-22,amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113. In yet otheraspects of this embodiment, a BAM22 binding domain comprises apolypeptide having, e.g., at most one, two, three, four or fivecontiguous amino acid deletions relative to amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; aminoacids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 ofSEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 113. In still other aspects of thisembodiment, a BAM22 binding domain comprises a polypeptide having, e.g.,at least one, two, three, four or five contiguous amino acid additionsrelative to amino acids 1-12, amino acids 6-22, amino acids 8-22 oramino acids 1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22,amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ IDNO: 110; amino acids 1-12, amino acids 6-22, amino acids 8-22 or aminoacids 1-22 of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, aminoacids 8-22 or amino acids 1-22 of SEQ ID NO: 112; or amino acids 1-12,amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO:113. In yet other aspects of this embodiment, a BAM22 binding domaincomprises a polypeptide having, e.g., at most one, two, three, four orfive contiguous amino acid additions relative to amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 108;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 111; aminoacids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 ofSEQ ID NO: 112; or amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 113.

In another embodiment, an opioid binding domain comprises an endomorphinpeptide. In another embodiment, an opioid binding domain is derived froman endomorphin peptide. In aspects of this embodiment, an endomorphinbinding domain comprises an endomorphin-1 or an endomorphin-2. In otheraspects of this embodiment, an endomorphin binding domain comprises SEQID NO: 114 or SEQ ID NO: 115.

In other aspects of this embodiment, an endomorphin binding domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith SEQ ID NO: 114 or SEQ ID NO: 115, at least 75% amino acid identitywith SEQ ID NO: 114 or SEQ ID NO: 115, at least 80% amino acid identitywith SEQ ID NO: 114 or SEQ ID NO: 115, at least 85% amino acid identitywith SEQ ID NO: 114 or SEQ ID NO: 115, at least 90% amino acid identitywith SEQ ID NO: 114 or SEQ ID NO: 115 or at least 95% amino acididentity with SEQ ID NO: 114 or SEQ ID NO: 115. In yet other aspects ofthis embodiment, an endomorphin binding domain comprises a polypeptidehaving, e.g., at most 70% amino acid identity with SEQ ID NO: 114 or SEQID NO: 115, at most 75% amino acid identity with SEQ ID NO: 114 or SEQID NO: 115, at most 80% amino acid identity with SEQ ID NO: 114 or SEQID NO: 115, at most 85% amino acid identity with SEQ ID NO: 114 or SEQID NO: 115, at most 90% amino acid identity with SEQ ID NO: 114 or SEQID NO: 115 or at most 95% amino acid identity with SEQ ID NO: 114 or SEQID NO: 115.

In other aspects of this embodiment, an endomorphin binding domaincomprises a polypeptide having, e.g., at least one, two or threenon-contiguous amino acid substitutions relative to SEQ ID NO: 114 orSEQ ID NO: 115. In other aspects of this embodiment, an endomorphinbinding domain comprises a polypeptide having, e.g., at most one, two orthree non-contiguous amino acid substitutions relative to SEQ ID NO: 114or SEQ ID NO: 115. In yet other aspects of this embodiment, anendomorphin binding domain comprises a polypeptide having, e.g., atleast one, two or three non-contiguous amino acid deletions relative toSEQ ID NO: 114 or SEQ ID NO: 115. In yet other aspects of thisembodiment, an endomorphin binding domain comprises a polypeptidehaving, e.g., at most one, two or three non-contiguous amino aciddeletions relative to SEQ ID NO: 114 or SEQ ID NO: 115. In still otheraspects of this embodiment, an endomorphin binding domain comprises apolypeptide having, e.g., at least one, two or three non-contiguousamino acid additions relative to SEQ ID NO: 114 or SEQ ID NO: 115. Inyet other aspects of this embodiment, an endomorphin binding domaincomprises a polypeptide having, e.g., at most one, two or threenon-contiguous amino acid additions relative to SEQ ID NO: 114 or SEQ IDNO: 115.

In other aspects of this embodiment, an endomorphin binding domaincomprises a polypeptide having, e.g., at least one, two or threecontiguous amino acid substitutions relative to SEQ ID NO: 114 or SEQ IDNO: 115. In other aspects of this embodiment, an endomorphin bindingdomain comprises a polypeptide having, e.g., at most one, two or threecontiguous amino acid substitutions relative to SEQ ID NO: 114 or SEQ IDNO: 115. In yet other aspects of this embodiment, an endomorphin bindingdomain comprises a polypeptide having, e.g., at least one, two or threecontiguous amino acid deletions relative to SEQ ID NO: 114 or SEQ ID NO:115. In yet other aspects of this embodiment, an endomorphin bindingdomain comprises a polypeptide having, e.g., at most one, two or threecontiguous amino acid deletions relative to SEQ ID NO: 114 or SEQ ID NO:115. In still other aspects of this embodiment, an endomorphin bindingdomain comprises a polypeptide having, e.g., at least one, two or threecontiguous amino acid additions relative to SEQ ID NO: 114 or SEQ ID NO:115. In yet other aspects of this embodiment, an endomorphin bindingdomain comprises a polypeptide having, e.g., at most one, two or threecontiguous amino acid additions relative to SEQ ID NO: 114 or SEQ ID NO:115.

In another embodiment, an opioid binding domain comprises an endorphinpeptide. In aspects of this embodiment, an opioid binding domaincomprises is derived from an endorphin peptide. In other aspects, anendorphin binding domain comprises an endorphin-α, a neoendorphin-α, anendorphin-β, a neoendorphin-β or an endorphin-γ. In other aspects ofthis embodiment, an endorphin binding domain comprises SEQ ID NO: 291,SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ IDNO: 120.

In other aspects of this embodiment, an endorphin binding domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQID NO: 119 or SEQ ID NO: 120, at least 75% amino acid identity with SEQID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO:119 or SEQ ID NO: 120, at least 80% amino acid identity with SEQ ID NO:291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 orSEQ ID NO: 120, at least 85% amino acid identity with SEQ ID NO: 291,SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ IDNO: 120, at least 90% amino acid identity with SEQ ID NO: 291, SEQ IDNO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO:120 or at least 95% amino acid identity with SEQ ID NO: 291, SEQ ID NO:116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120.In yet other aspects of this embodiment, an endorphin binding domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQID NO: 119 or SEQ ID NO: 120, at most 75% amino acid identity with SEQID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO:119 or SEQ ID NO: 120, at most 80% amino acid identity with SEQ ID NO:291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 orSEQ ID NO: 120, at most 85% amino acid identity with SEQ ID NO: 291, SEQID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO:120, at most 90% amino acid identity with SEQ ID NO: 291, SEQ ID NO:116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120 orat most 95% amino acid identity with SEQ ID NO: 291, SEQ ID NO: 116, SEQID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120.

In other aspects of this embodiment, an endorphin binding domaincomprises a polypeptide having, e.g., at least one, two, three, four orfive non-contiguous amino acid substitutions relative to SEQ ID NO: 291,SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ IDNO: 120. In other aspects of this embodiment, an endorphin bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four or five non-contiguous amino acid substitutions relative to SEQ IDNO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119or SEQ ID NO: 120. In yet other aspects of this embodiment, an endorphinbinding domain comprises a polypeptide having, e.g., at least one, two,three, four or five non-contiguous amino acid deletions relative to SEQID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO:119 or SEQ ID NO: 120. In yet other aspects of this embodiment, anendorphin binding domain comprises a polypeptide having, e.g., at mostone, two, three, four or five non-contiguous amino acid deletionsrelative to SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO:118, SEQ ID NO: 119 or SEQ ID NO: 120. In still other aspects of thisembodiment, an endorphin binding domain comprises a polypeptide having,e.g., at least one, two, three, four or five non-contiguous amino acidadditions relative to SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117,SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120. In yet other aspectsof this embodiment, an endorphin binding domain comprises a polypeptidehaving, e.g., at most one, two, three, four or five non-contiguous aminoacid additions relative to SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO:117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120.

In other aspects of this embodiment, an endorphin binding domaincomprises a polypeptide having, e.g., at least one, two, three, four orfive contiguous amino acid substitutions relative to SEQ ID NO: 291, SEQID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO:120. In other aspects of this embodiment, an endorphin binding domaincomprises a polypeptide having, e.g., at most one, two, three, four orfive contiguous amino acid substitutions relative to SEQ ID NO: 291, SEQID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO:120. In yet other aspects of this embodiment, an endorphin bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four or five contiguous amino acid deletions relative to SEQ ID NO: 291,SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ IDNO: 120. In yet other aspects of this embodiment, an endorphin bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four or five contiguous amino acid deletions relative to SEQ ID NO: 291,SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ IDNO: 120. In still other aspects of this embodiment, an endorphin bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four or five contiguous amino acid additions relative to SEQ ID NO: 291,SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ IDNO: 120. In yet other aspects of this embodiment, an endorphin bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four or five contiguous amino acid additions relative to SEQ ID NO: 291,SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ IDNO: 120.

In another embodiment, an opioid binding domain comprises a dynorphinpeptide. In another embodiment, an opioid binding domain is derived froma dynorphin peptide. In aspects of this embodiment, a dynorphin bindingdomain comprises a dynorphin A, a dynorphin B (leumorphin) or arimorphin. In other aspects of this embodiment, a dynorphin bindingdomain comprises SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ IDNO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128,SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ IDNO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137,SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ IDNO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146,SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150 or SEQ IDNO: 151.

In other aspects of this embodiment, a dynorphin binding domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146, at least 75%amino acid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO:146, at least 80% amino acid identity with SEQ ID NO: 121, SEQ ID NO:130 or SEQ ID NO: 146, at least 85% amino acid identity with SEQ ID NO:121, SEQ ID NO: 130 or SEQ ID NO: 146, at least 90% amino acid identitywith SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146 or at least 95%amino acid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO:146. In yet other aspects of this embodiment, a dynorphin binding domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146, at most 75% aminoacid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146, atmost 80% amino acid identity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQID NO: 146, at most 85% amino acid identity with SEQ ID NO: 121, SEQ IDNO: 130 or SEQ ID NO: 146, at most 90% amino acid identity with SEQ IDNO: 121, SEQ ID NO: 130 or SEQ ID NO: 146 or at most 95% amino acididentity with SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146.

In other aspects of this embodiment, a dynorphin binding domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten non-contiguous amino acidsubstitutions relative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO:146. In other aspects of this embodiment, a dynorphin binding domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or ten non-contiguous amino acidsubstitutions relative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO:146. In yet other aspects of this embodiment, a dynorphin binding domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten non-contiguous amino acid deletionsrelative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In yetother aspects of this embodiment, a dynorphin binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid deletions relativeto SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In still otheraspects of this embodiment, a dynorphin binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid additions relativeto SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In yet otheraspects of this embodiment, a dynorphin binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid additions relativeto SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146.

In other aspects of this embodiment, a dynorphin binding domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten contiguous amino acid substitutionsrelative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In otheraspects of this embodiment, a dynorphin binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten contiguous amino acid substitutions relativeto SEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In yet otheraspects of this embodiment, a dynorphin binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or ten contiguous amino acid deletions relative toSEQ ID NO: 121, SEQ ID NO: 130 or SEQ ID NO: 146. In yet other aspectsof this embodiment, a dynorphin binding domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine or ten contiguous amino acid deletions relative to SEQ ID NO: 121,SEQ ID NO: 130 or SEQ ID NO: 146. In still other aspects of thisembodiment, a dynorphin binding domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine orten contiguous amino acid additions relative to SEQ ID NO: 121, SEQ IDNO: 130 or SEQ ID NO: 146. In yet other aspects of this embodiment, adynorphin binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or ten contiguousamino acid additions relative to SEQ ID NO: 121, SEQ ID NO: 130 or SEQID NO: 146.

In another embodiment, an opioid binding domain comprises a nociceptinpeptide. In another embodiment, an opioid binding domain is derived froma nociceptin peptide. In aspects of this embodiment, a nociceptinbinding domain comprises a nociceptin RK, a nociceptin, a neuropeptide1, a neuropeptide 2 or a neuropeptide 3. In other aspects of thisembodiment, a nociceptin binding domain comprises SEQ ID NO: 152, SEQ IDNO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157,SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161.

In other aspects of this embodiment, a nociceptin binding domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161,at least 75% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159,SEQ ID NO: 160 or SEQ ID NO: 161, at least 80% amino acid identity withSEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161, atleast 85% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQID NO: 160 or SEQ ID NO: 161, at least 90% amino acid identity with SEQID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161 or at least95% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO:160 or SEQ ID NO: 161. In yet other aspects of this embodiment, anociceptin binding domain comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO:160 or SEQ ID NO: 161, at most 75% amino acid identity with SEQ ID NO:152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161, at most 80% aminoacid identity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQID NO: 161, at most 85% amino acid identity with SEQ ID NO: 152, SEQ IDNO: 159, SEQ ID NO: 160 or SEQ ID NO: 161, at most 90% amino acididentity with SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ IDNO: 161 or at most 95% amino acid identity with SEQ ID NO: 152, SEQ IDNO: 159, SEQ ID NO: 160 or SEQ ID NO: 161.

In other aspects of this embodiment, a nociceptin binding domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten non-contiguous amino acidsubstitutions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160or SEQ ID NO: 161. In other aspects of this embodiment, a nociceptinbinding domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid substitutions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ IDNO: 160 or SEQ ID NO: 161. In yet other aspects of this embodiment, anociceptin binding domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid deletions relative to SEQ ID NO: 152, SEQ IDNO: 159, SEQ ID NO: 160 or SEQ ID NO: 161. In yet other aspects of thisembodiment, a nociceptin binding domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine orten non-contiguous amino acid deletions relative to SEQ ID NO: 152, SEQID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161. In still other aspects ofthis embodiment, a nociceptin binding domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine or ten non-contiguous amino acid additions relative to SEQ ID NO:152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161. In yet otheraspects of this embodiment, a nociceptin binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid additions relativeto SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO: 161.

In other aspects of this embodiment, a nociceptin binding domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten contiguous amino acid substitutionsrelative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO:161. In other aspects of this embodiment, a nociceptin binding domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or ten contiguous amino acid substitutionsrelative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 or SEQ ID NO:161. In yet other aspects of this embodiment, a nociceptin bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or ten contiguous amino aciddeletions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 orSEQ ID NO: 161. In yet other aspects of this embodiment, a nociceptinbinding domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine or ten contiguous amino aciddeletions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 orSEQ ID NO: 161. In still other aspects of this embodiment, a nociceptinbinding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 orSEQ ID NO: 161. In yet other aspects of this embodiment, a nociceptinbinding domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 152, SEQ ID NO: 159, SEQ ID NO: 160 orSEQ ID NO: 161.

Another example of a binding domain disclosed in the presentspecification is, e.g., a melanocortin peptide, such as, e.g., amelanocyte stimulating hormone, an adrenocorticotropin, aCorticotropin-like intermediary peptide) or a lipotropin. Thus, in anembodiment, a binding domain is derived from a melanocortin peptide.

In another embodiment, a binding domain comprises a melanocortinpeptide. In another embodiment, a binding domain is derived from amelanocortin peptide. In an aspect of this embodiment, a melanocortinpeptide binding domain comprises a melanocyte stimulating hormone. In anaspect of this embodiment, a melanocortin peptide binding domaincomprises is derived from a melanocyte stimulating hormone. In aspectsof this embodiment, a melanocyte stimulating hormone binding domaincomprises an α-melanocyte stimulating hormones (α-MSH), a β-melanocytestimulating hormones (β-MSH), a γ-melanocyte stimulating hormones(γ-MSH). In other aspects of this embodiment, a melanocyte stimulatinghormone binding domain comprises SEQ ID NO: 162, SEQ ID NO: 163 or SEQID NO: 164.

In other aspects of this embodiment, a melanocyte stimulating hormonebinding domain comprises a polypeptide having, e.g., at least 70% aminoacid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164, atleast 75% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQID NO: 164, at least 80% amino acid identity with SEQ ID NO: 162, SEQ IDNO: 163 or SEQ ID NO: 164, at least 85% amino acid identity with SEQ IDNO: 162, SEQ ID NO: 163 or SEQ ID NO: 164, at least 90% amino acididentity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164 or atleast 95% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQID NO: 164. In yet other aspects of this embodiment, a melanocytestimulating hormone binding domain comprises a polypeptide having, e.g.,at most 70% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 orSEQ ID NO: 164, at most 75% amino acid identity with SEQ ID NO: 162, SEQID NO: 163 or SEQ ID NO: 164, at most 80% amino acid identity with SEQID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164, at most 85% amino acididentity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164, at most90% amino acid identity with SEQ ID NO: 162, SEQ ID NO: 163 or SEQ IDNO: 164 or at most 95% amino acid identity with SEQ ID NO: 162, SEQ IDNO: 163 or SEQ ID NO: 164.

In other aspects of this embodiment, a melanocyte stimulating hormonebinding domain comprises a polypeptide having, e.g., at least one, two,three, four or five non-contiguous amino acid substitutions relative toSEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164. In other aspects ofthis embodiment, a melanocyte stimulating hormone binding domaincomprises a polypeptide having, e.g., at most one, two, three, four orfive non-contiguous amino acid substitutions relative to SEQ ID NO: 162,SEQ ID NO: 163 or SEQ ID NO: 164. In yet other aspects of thisembodiment, a melanocyte stimulating hormone binding domain comprises apolypeptide having, e.g., at least one, two, three, four or fivenon-contiguous amino acid deletions relative to SEQ ID NO: 162, SEQ IDNO: 163 or SEQ ID NO: 164. In yet other aspects of this embodiment, amelanocyte stimulating hormone binding domain comprises a polypeptidehaving, e.g., at most one, two, three, four or five non-contiguous aminoacid deletions relative to SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO:164. In still other aspects of this embodiment, a melanocyte stimulatinghormone binding domain comprises a polypeptide having, e.g., at leastone, two, three, four or five non-contiguous amino acid additionsrelative to SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164. In yetother aspects of this embodiment, a melanocyte stimulating hormonebinding domain comprises a polypeptide having, e.g., at most one, two,three, four or five non-contiguous amino acid additions relative to SEQID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164.

In other aspects of this embodiment, a melanocyte stimulating hormonebinding domain comprises a polypeptide having, e.g., at least one, two,three, four or five contiguous amino acid substitutions relative to SEQID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164. In other aspects of thisembodiment, a melanocyte stimulating hormone binding domain comprises apolypeptide having, e.g., at most one, two, three, four or fivecontiguous amino acid substitutions relative to SEQ ID NO: 162, SEQ IDNO: 163 or SEQ ID NO: 164. In yet other aspects of this embodiment, amelanocyte stimulating hormone binding domain comprises a polypeptidehaving, e.g., at least one, two, three, four or five contiguous aminoacid deletions relative to SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO:164. In yet other aspects of this embodiment, a melanocyte stimulatinghormone binding domain comprises a polypeptide having, e.g., at mostone, two, three, four or five contiguous amino acid deletions relativeto SEQ ID NO: 162, SEQ ID NO: 163 or SEQ ID NO: 164. In still otheraspects of this embodiment, a melanocyte stimulating hormone bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four or five contiguous amino acid additions relative to SEQ ID NO: 162,SEQ ID NO: 163 or SEQ ID NO: 164. In yet other aspects of thisembodiment, a melanocyte stimulating hormone binding domain comprises apolypeptide having, e.g., at most one, two, three, four or fivecontiguous amino acid additions relative to SEQ ID NO: 162, SEQ ID NO:163 or SEQ ID NO: 164.

In another embodiment, a melanocortin peptide binding domain comprisesan adrenocorticotropin. In another embodiment, a melanocortin peptidebinding domain is derived from an adrenocorticotropin. In aspects ofthis embodiment, an adrenocorticotropin binding domain comprises anadrenocorticotropin (ACTH) or a Corticotropin-like intermediary peptide(CLIP). In other aspects of this embodiment, an adrenocorticotropinbinding domain comprises SEQ ID NO: 165 or SEQ ID NO: 166.

In other aspects of this embodiment, an adrenocorticotropin bindingdomain comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 165 or SEQ ID NO: 166, at least 75% amino acididentity with SEQ ID NO: 165 or SEQ ID NO: 166, at least 80% amino acididentity with SEQ ID NO: 165 or SEQ ID NO: 166, at least 85% amino acididentity with SEQ ID NO: 165 or SEQ ID NO: 166, at least 90% amino acididentity with SEQ ID NO: 165 or SEQ ID NO: 166 or at least 95% aminoacid identity with SEQ ID NO: 165 or SEQ ID NO: 166. In yet otheraspects of this embodiment, an adrenocorticotropin binding domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith SEQ ID NO: 165 or SEQ ID NO: 166, at most 75% amino acid identitywith SEQ ID NO: 165 or SEQ ID NO: 166, at most 80% amino acid identitywith SEQ ID NO: 165 or SEQ ID NO: 166, at most 85% amino acid identitywith SEQ ID NO: 165 or SEQ ID NO: 166, at most 90% amino acid identitywith SEQ ID NO: 165 or SEQ ID NO: 166 or at most 95% amino acid identitywith SEQ ID NO: 165 or SEQ ID NO: 166.

In other aspects of this embodiment, an adrenocorticotropin bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four or five non-contiguous amino acid substitutions relative to SEQ IDNO: 165 or SEQ ID NO: 166. In other aspects of this embodiment, anadrenocorticotropin binding domain comprises a polypeptide having, e.g.,at most one, two, three, four or five non-contiguous amino acidsubstitutions relative to SEQ ID NO: 165 or SEQ ID NO: 166. In yet otheraspects of this embodiment, an adrenocorticotropin binding domaincomprises a polypeptide having, e.g., at least one, two, three, four orfive non-contiguous amino acid deletions relative to SEQ ID NO: 165 orSEQ ID NO: 166. In yet other aspects of this embodiment, anadrenocorticotropin binding domain comprises a polypeptide having, e.g.,at most one, two, three, four or five non-contiguous amino aciddeletions relative to SEQ ID NO: 165 or SEQ ID NO: 166. In still otheraspects of this embodiment, an adrenocorticotropin binding domaincomprises a polypeptide having, e.g., at least one, two, three, four orfive non-contiguous amino acid additions relative to SEQ ID NO: 165 orSEQ ID NO: 166. In yet other aspects of this embodiment, anadrenocorticotropin binding domain comprises a polypeptide having, e.g.,at most one, two, three, four or five non-contiguous amino acidadditions relative to SEQ ID NO: 165 or SEQ ID NO: 166.

In other aspects of this embodiment, an adrenocorticotropin bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four or five contiguous amino acid substitutions relative to SEQ ID NO:165 or SEQ ID NO: 166. In other aspects of this embodiment, anadrenocorticotropin binding domain comprises a polypeptide having, e.g.,at most one, two, three, four or five contiguous amino acidsubstitutions relative to SEQ ID NO: 165 or SEQ ID NO: 166. In yet otheraspects of this embodiment, an adrenocorticotropin binding domaincomprises a polypeptide having, e.g., at least one, two, three, four orfive contiguous amino acid deletions relative to SEQ ID NO: 165 or SEQID NO: 166. In yet other aspects of this embodiment, anadrenocorticotropin binding domain comprises a polypeptide having, e.g.,at most one, two, three, four or five contiguous amino acid deletionsrelative to SEQ ID NO: 165 or SEQ ID NO: 166. In still other aspects ofthis embodiment, an adrenocorticotropin binding domain comprises apolypeptide having, e.g., at least one, two, three, four or fivecontiguous amino acid additions relative to SEQ ID NO: 165 or SEQ ID NO:166. In yet other aspects of this embodiment, an adrenocorticotropinbinding domain comprises a polypeptide having, e.g., at most one, two,three, four or five contiguous amino acid additions relative to SEQ IDNO: 165 or SEQ ID NO: 166.

In another embodiment, a melanocortin peptide binding domain comprises alipotropin. In another embodiment, a melanocortin peptide binding domainis derived from a lipotropin. In aspects of this embodiment, alipotropin binding domain comprises a β-lipotropin (β-LPH) or aγ-lipotropin (γ-LPH). In other aspects of this embodiment, a lipotropinbinding domain comprises SEQ ID NO: 167 or SEQ ID NO: 168.

In other aspects of this embodiment, a lipotropin binding domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith SEQ ID NO: 167 or SEQ ID NO: 168, at least 75% amino acid identitywith SEQ ID NO: 167 or SEQ ID NO: 168, at least 80% amino acid identitywith SEQ ID NO: 167 or SEQ ID NO: 168, at least 85% amino acid identitywith SEQ ID NO: 167 or SEQ ID NO: 168, at least 90% amino acid identitywith SEQ ID NO: 167 or SEQ ID NO: 168 or at least 95% amino acididentity with SEQ ID NO: 167 or SEQ ID NO: 168. In yet other aspects ofthis embodiment, a lipotropin binding domain comprises a polypeptidehaving, e.g., at most 70% amino acid identity with SEQ ID NO: 167 or SEQID NO: 168, at most 75% amino acid identity with SEQ ID NO: 167 or SEQID NO: 168, at most 80% amino acid identity with SEQ ID NO: 167 or SEQID NO: 168, at most 85% amino acid identity with SEQ ID NO: 167 or SEQID NO: 168, at most 90% amino acid identity with SEQ ID NO: 167 or SEQID NO: 168 or at most 95% amino acid identity with SEQ ID NO: 167 or SEQID NO: 168.

In other aspects of this embodiment, a lipotropin binding domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten non-contiguous amino acidsubstitutions relative to SEQ ID NO: 167 or SEQ ID NO: 168. In otheraspects of this embodiment, a lipotropin binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid substitutionsrelative to SEQ ID NO: 167 or SEQ ID NO: 168. In yet other aspects ofthis embodiment, a lipotropin binding domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine or ten non-contiguous amino acid deletions relative to SEQ ID NO:167 or SEQ ID NO: 168. In yet other aspects of this embodiment, alipotropin binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid deletions relative to SEQ ID NO: 167 or SEQ IDNO: 168. In still other aspects of this embodiment, a lipotropin bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or ten non-contiguous amino acidadditions relative to SEQ ID NO: 167 or SEQ ID NO: 168. In yet otheraspects of this embodiment, a lipotropin binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid additions relativeto SEQ ID NO: 167 or SEQ ID NO: 168.

In other aspects of this embodiment, a lipotropin binding domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten contiguous amino acid substitutionsrelative to SEQ ID NO: 167 or SEQ ID NO: 168. In other aspects of thisembodiment, a lipotropin binding domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine orten contiguous amino acid substitutions relative to SEQ ID NO: 167 orSEQ ID NO: 168. In yet other aspects of this embodiment, a lipotropinbinding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or ten contiguous amino aciddeletions relative to SEQ ID NO: 167 or SEQ ID NO: 168. In yet otheraspects of this embodiment, a lipotropin binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten contiguous amino acid deletions relative toSEQ ID NO: 167 or SEQ ID NO: 168. In still other aspects of thisembodiment, a lipotropin binding domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine orten contiguous amino acid additions relative to SEQ ID NO: 167 or SEQ IDNO: 168. In yet other aspects of this embodiment, a lipotropin bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 167 or SEQ ID NO: 168.

In another embodiment, a melanocortin peptide binding domain comprises aneuropeptide derived from a melanocortin peptide. In another embodiment,a melanocortin peptide binding domain is derived from a neuropeptidederived from a melanocortin peptide. In aspects of this embodiment, amelanocortin peptide derived neuropeptide binding domain comprises SEQID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171.

In other aspects of this embodiment, a melanocortin peptide derivedneuropeptide binding domain comprises a polypeptide having, e.g., atleast 70% amino acid identity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQID NO: 171, at least 75% amino acid identity with SEQ ID NO: 169, SEQ IDNO: 170 or SEQ ID NO: 171, at least 80% amino acid identity with SEQ IDNO: 169, SEQ ID NO: 170 or SEQ ID NO: 171, at least 85% amino acididentity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171, at least90% amino acid identity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ IDNO: 171 or at least 95% amino acid identity with SEQ ID NO: 169, SEQ IDNO: 170 or SEQ ID NO: 171. In yet other aspects of this embodiment, amelanocortin peptide derived neuropeptide binding domain comprises apolypeptide having, e.g., at most 70% amino acid identity with SEQ IDNO: 169, SEQ ID NO: 170 or SEQ ID NO: 171, at most 75% amino acididentity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171, at most80% amino acid identity with SEQ ID NO: 169, SEQ ID NO: 170 or SEQ IDNO: 171, at most 85% amino acid identity with SEQ ID NO: 169, SEQ ID NO:170 or SEQ ID NO: 171, at most 90% amino acid identity with SEQ ID NO:169, SEQ ID NO: 170 or SEQ ID NO: 171 or at most 95% amino acid identitywith SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171.

In other aspects of this embodiment, a melanocortin peptide derivedneuropeptide binding domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid substitutions relative to SEQ ID NO: 169, SEQID NO: 170 or SEQ ID NO: 171. In other aspects of this embodiment, amelanocortin peptide derived neuropeptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid substitutionsrelative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171. In yetother aspects of this embodiment, a melanocortin peptide derivedneuropeptide binding domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid deletions relative to SEQ ID NO: 169, SEQ IDNO: 170 or SEQ ID NO: 171. In yet other aspects of this embodiment, amelanocortin peptide derived neuropeptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid deletions relativeto SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171. In still otheraspects of this embodiment, a melanocortin peptide derived neuropeptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid additions relative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO:171. In yet other aspects of this embodiment, a melanocortin peptidederived neuropeptide binding domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine orten non-contiguous amino acid additions relative to SEQ ID NO: 169, SEQID NO: 170 or SEQ ID NO: 171.

In other aspects of this embodiment, a melanocortin peptide derivedneuropeptide binding domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine or tencontiguous amino acid substitutions relative to SEQ ID NO: 169, SEQ IDNO: 170 or SEQ ID NO: 171. In other aspects of this embodiment, amelanocortin peptide derived neuropeptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten contiguous amino acid substitutions relativeto SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171. In yet otheraspects of this embodiment, a melanocortin peptide derived neuropeptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or ten contiguous amino aciddeletions relative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171.In yet other aspects of this embodiment, a melanocortin peptide derivedneuropeptide binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine or tencontiguous amino acid deletions relative to SEQ ID NO: 169, SEQ ID NO:170 or SEQ ID NO: 171. In still other aspects of this embodiment, amelanocortin peptide derived neuropeptide binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or ten contiguous amino acid additions relative toSEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171. In yet other aspectsof this embodiment, a melanocortin peptide derived neuropeptide bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 169, SEQ ID NO: 170 or SEQ ID NO: 171.

In another embodiment, a binding domain comprises a galanin. In anotherembodiment, a binding domain is derived from a galanin. In aspects ofthis embodiment, a galanin binding domain comprises a galanin or agalanin message-associated peptide (GMAP). In other aspects of thisembodiment, a galanin binding domain comprises SEQ ID NO: 172 or SEQ IDNO: 173.

In other aspects of this embodiment, a galanin binding domain comprisesa polypeptide having, e.g., at least 70% amino acid identity with SEQ IDNO: 172 or SEQ ID NO: 173, at least 75% amino acid identity with SEQ IDNO: 172 or SEQ ID NO: 173, at least 80% amino acid identity with SEQ IDNO: 172 or SEQ ID NO: 173, at least 85% amino acid identity with SEQ IDNO: 172 or SEQ ID NO: 173, at least 90% amino acid identity with SEQ IDNO: 172 or SEQ ID NO: 173 or at least 95% amino acid identity with SEQID NO: 172 or SEQ ID NO: 173. In yet other aspects of this embodiment, agalanin binding domain comprises a polypeptide having, e.g., at most 70%amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173, at most 75%amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173, at most 80%amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173, at most 85%amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173, at most 90%amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173 or at most 95%amino acid identity with SEQ ID NO: 172 or SEQ ID NO: 173.

In other aspects of this embodiment, a galanin binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid substitutionsrelative to SEQ ID NO: 172 or SEQ ID NO: 173. In other aspects of thisembodiment, a galanin binding domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine orten non-contiguous amino acid substitutions relative to SEQ ID NO: 172or SEQ ID NO: 173. In yet other aspects of this embodiment, a galaninbinding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid deletions relative to SEQ ID NO: 172 or SEQ ID NO: 173. In yetother aspects of this embodiment, a galanin binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid deletions relativeto SEQ ID NO: 172 or SEQ ID NO: 173. In still other aspects of thisembodiment, a galanin binding domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine orten non-contiguous amino acid additions relative to SEQ ID NO: 172 orSEQ ID NO: 173. In yet other aspects of this embodiment, a galaninbinding domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid additions relative to SEQ ID NO: 172 or SEQ ID NO: 173.

In other aspects of this embodiment, a galanin binding domain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or ten contiguous amino acid substitutions relativeto SEQ ID NO: 172 or SEQ ID NO: 173. In other aspects of thisembodiment, a galanin binding domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine orten contiguous amino acid substitutions relative to SEQ ID NO: 172 orSEQ ID NO: 173. In yet other aspects of this embodiment, a galaninbinding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or ten contiguous amino aciddeletions relative to SEQ ID NO: 172 or SEQ ID NO: 173. In yet otheraspects of this embodiment, a galanin binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten contiguous amino acid deletions relative toSEQ ID NO: 172 or SEQ ID NO: 173. In still other aspects of thisembodiment, a galanin binding domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine orten contiguous amino acid additions relative to SEQ ID NO: 172 or SEQ IDNO: 173. In yet other aspects of this embodiment, a galanin bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 172 or SEQ ID NO: 173.

Another example of a binding domain disclosed in the presentspecification is, e.g., a granin peptide, such as, e.g., a chromograninA, a chromogranin B (secretogranin 1) or a chromogranin C (secretograninII). Thus, in an embodiment, a binding domain comprises a graninpeptide. In another embodiment, a binding domain is derived from agranin peptide.

In another embodiment, a granin peptide binding domain comprises achromogranin A peptide. In another embodiment, a granin peptide bindingdomain is derived from a chromogranin A peptide. In aspects of thisembodiment, a chromogranin A peptide binding domain comprises aβ-granin, a vasostatin, a chromostatin, a pancreastatin, a WE-14, acatestatin, a parastatin or a GE-25. In other aspects of thisembodiment, a chromogranin A peptide binding domain comprises SEQ ID NO:174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181.

In other aspects of this embodiment, a chromogranin A peptide bindingdomain comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO:177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181,at least 75% amino acid identity with SEQ ID NO: 174, SEQ ID NO: 175,SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ IDNO: 180 or SEQ ID NO: 181, at least 80% amino acid identity with SEQ IDNO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178,SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181, at least 85% aminoacid identity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO:181, at least 90% amino acid identity with SEQ ID NO: 174, SEQ ID NO:175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQID NO: 180 or SEQ ID NO: 181 or at least 95% amino acid identity withSEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ IDNO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In yet otheraspects of this embodiment, a chromogranin A peptide binding domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181, at most75% amino acid identity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO:176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 orSEQ ID NO: 181, at most 80% amino acid identity with SEQ ID NO: 174, SEQID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO:179, SEQ ID NO: 180 or SEQ ID NO: 181, at most 85% amino acid identitywith SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181, at most90% amino acid identity with SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO:176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 orSEQ ID NO: 181 or at most 95% amino acid identity with SEQ ID NO: 174,SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ IDNO: 179, SEQ ID NO: 180 or SEQ ID NO: 181.

In other aspects of this embodiment, a chromogranin A peptide bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or ten non-contiguous amino acidsubstitutions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO:176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 orSEQ ID NO: 181. In other aspects of this embodiment, a chromogranin Apeptide binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid substitutions relative to SEQ ID NO: 174, SEQID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO:179, SEQ ID NO: 180 or SEQ ID NO: 181. In yet other aspects of thisembodiment, a chromogranin A peptide binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid deletions relativeto SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In yetother aspects of this embodiment, a chromogranin A peptide bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten non-contiguous amino aciddeletions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176,SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ IDNO: 181. In still other aspects of this embodiment, a chromogranin Apeptide binding domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid additions relative to SEQ ID NO: 174, SEQ IDNO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179,SEQ ID NO: 180 or SEQ ID NO: 181. In yet other aspects of thisembodiment, a chromogranin A peptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid additions relativeto SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181.

In other aspects of this embodiment, a chromogranin A peptide bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or ten contiguous amino acidsubstitutions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO:176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 orSEQ ID NO: 181. In other aspects of this embodiment, a chromogranin Apeptide binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or ten contiguousamino acid substitutions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO:180 or SEQ ID NO: 181. In yet other aspects of this embodiment, achromogranin A peptide binding domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine orten contiguous amino acid deletions relative to SEQ ID NO: 174, SEQ IDNO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179,SEQ ID NO: 180 or SEQ ID NO: 181. In yet other aspects of thisembodiment, a chromogranin A peptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten contiguous amino acid deletions relative toSEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ IDNO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181. In stillother aspects of this embodiment, a chromogranin A peptide bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176,SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ IDNO: 181. In yet other aspects of this embodiment, a chromogranin Apeptide binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or ten contiguousamino acid additions relative to SEQ ID NO: 174, SEQ ID NO: 175, SEQ IDNO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180or SEQ ID NO: 181.

In another embodiment, a granin peptide binding domain comprises achromogranin B peptide. In another embodiment, a granin peptide bindingdomain is derived from a chromogranin B peptide. In aspects of thisembodiment, a chromogranin B peptide binding domain comprises a GAWKpeptide, an adrenomedullary peptide or a secretolytin. In other aspectsof this embodiment, a chromogranin B peptide binding domain comprisesSEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ IDNO: 186.

In other aspects of this embodiment, a chromogranin B peptide bindingdomain comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO:185 or SEQ ID NO: 186, at least 75% amino acid identity with SEQ ID NO:182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186,at least 80% amino acid identity with SEQ ID NO: 182, SEQ ID NO: 183,SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186, at least 85% aminoacid identity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQID NO: 185 or SEQ ID NO: 186, at least 90% amino acid identity with SEQID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO:186 or at least 95% amino acid identity with SEQ ID NO: 182, SEQ ID NO:183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186. In yet otheraspects of this embodiment, a chromogranin B peptide binding domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 orSEQ ID NO: 186, at most 75% amino acid identity with SEQ ID NO: 182, SEQID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186, at most80% amino acid identity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO:184, SEQ ID NO: 185 or SEQ ID NO: 186, at most 85% amino acid identitywith SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 orSEQ ID NO: 186, at most 90% amino acid identity with SEQ ID NO: 182, SEQID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186 or at most95% amino acid identity with SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO:184, SEQ ID NO: 185 or SEQ ID NO: 186.

In other aspects of this embodiment, a chromogranin B peptide bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or ten non-contiguous amino acidsubstitutions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO:184, SEQ ID NO: 185 or SEQ ID NO: 186. In other aspects of thisembodiment, a chromogranin B peptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid substitutionsrelative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO:185 or SEQ ID NO: 186. In yet other aspects of this embodiment, achromogranin B peptide binding domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine orten non-contiguous amino acid deletions relative to SEQ ID NO: 182, SEQID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186. In yetother aspects of this embodiment, a chromogranin B peptide bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten non-contiguous amino aciddeletions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184,SEQ ID NO: 185 or SEQ ID NO: 186. In still other aspects of thisembodiment, a chromogranin B peptide binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid additions relativeto SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQID NO: 186. In yet other aspects of this embodiment, a chromogranin Bpeptide binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid additions relative to SEQ ID NO: 182, SEQ IDNO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 186.

In other aspects of this embodiment, a chromogranin B peptide bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or ten contiguous amino acidsubstitutions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO:184, SEQ ID NO: 185 or SEQ ID NO: 186. In other aspects of thisembodiment, a chromogranin B peptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten contiguous amino acid substitutions relativeto SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQID NO: 186. In yet other aspects of this embodiment, a chromogranin Bpeptide binding domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or ten contiguousamino acid deletions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ IDNO: 184, SEQ ID NO: 185 or SEQ ID NO: 186. In yet other aspects of thisembodiment, a chromogranin B peptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten contiguous amino acid deletions relative toSEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ IDNO: 186. In still other aspects of this embodiment, a chromogranin Bpeptide binding domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or ten contiguousamino acid additions relative to SEQ ID NO: 182, SEQ ID NO: 183, SEQ IDNO: 184, SEQ ID NO: 185 or SEQ ID NO: 186. In yet other aspects of thisembodiment, a chromogranin B peptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten contiguous amino acid additions relative toSEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ IDNO: 186.

In another embodiment, a granin peptide binding domain comprises achromogranin C peptide. In another embodiment, a granin peptide bindingdomain is derived from a chromogranin C peptide. In aspects of thisembodiment, a chromogranin C peptide binding domain comprises asecretoneurin. In other aspects of this embodiment, a chromogranin Cpeptide binding domain comprises SEQ ID NO: 187.

In other aspects of this embodiment, a chromogranin C peptide bindingdomain comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 187, at least 75% amino acid identity with SEQID NO: 187, at least 80% amino acid identity with SEQ ID NO: 187, atleast 85% amino acid identity with SEQ ID NO: 187, at least 90% aminoacid identity with SEQ ID NO: 187 or at least 95% amino acid identitywith SEQ ID NO: 187. In yet other aspects of this embodiment, achromogranin C peptide binding domain comprises a polypeptide having,e.g., at most 70% amino acid identity with SEQ ID NO: 187, at most 75%amino acid identity with SEQ ID NO: 187, at most 80% amino acid identitywith SEQ ID NO: 187, at most 85% amino acid identity with SEQ ID NO:187, at most 90% amino acid identity with SEQ ID NO: 187 or at most 95%amino acid identity with SEQ ID NO: 187.

In other aspects of this embodiment, a chromogranin C peptide bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or ten non-contiguous amino acidsubstitutions relative to SEQ ID NO: 187. In other aspects of thisembodiment, a chromogranin C peptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid substitutionsrelative to SEQ ID NO: 187. In yet other aspects of this embodiment, achromogranin C peptide binding domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine orten non-contiguous amino acid deletions relative to SEQ ID NO: 187. Inyet other aspects of this embodiment, a chromogranin C peptide bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten non-contiguous amino aciddeletions relative to SEQ ID NO: 187. In still other aspects of thisembodiment, a chromogranin C peptide binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid additions relativeto SEQ ID NO: 187. In yet other aspects of this embodiment, achromogranin C peptide binding domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine orten non-contiguous amino acid additions relative to SEQ ID NO: 187.

In other aspects of this embodiment, a chromogranin C peptide bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or ten contiguous amino acidsubstitutions relative to SEQ ID NO: 187. In other aspects of thisembodiment, a chromogranin C peptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten contiguous amino acid substitutions relativeto SEQ ID NO: 187. In yet other aspects of this embodiment, achromogranin C peptide binding domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine orten contiguous amino acid deletions relative to SEQ ID NO: 187. In yetother aspects of this embodiment, a chromogranin C peptide bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten contiguous amino aciddeletions relative to SEQ ID NO: 187. In still other aspects of thisembodiment, a chromogranin C peptide binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or ten contiguous amino acid additions relative toSEQ ID NO: 187. In yet other aspects of this embodiment, a chromograninC peptide binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or ten contiguousamino acid additions relative to SEQ ID NO: 187.

Another example of a binding domain disclosed in the presentspecification is, e.g., a tachykinin peptide, such as, e.g., a SubstanceP, a neuropeptide K (NPK), a neuropeptide gamma (NP gamma), a neurokininA (NKA; Substance K, neurokinin alpha, neuromedin L), a neurokinin B(NKB), a hemokinin or a endokinin. Thus, in an embodiment, a bindingdomain comprises a tachykinin peptide. In an embodiment, a bindingdomain is derived from a tachykinin peptide.

In aspects of this embodiment, a tachykinin peptide binding domaincomprises a Substance P, a neuropeptide K (NPK), a neuropeptide gamma(NP gamma), a neurokinin A (NKA; Substance K, neurokinin alpha,neuromedin L), a neurokinin B (NKB), a hemokinin or a endokinin. Inaspects of this embodiment, a tachykinin peptide binding domain isderived from a Substance P, a neuropeptide K (NPK), a neuropeptide gamma(NP gamma), a neurokinin A (NKA; Substance K, neurokinin alpha,neuromedin L), a neurokinin B (NKB), a hemokinin or a endokinin. Inother aspects of this embodiment, a tachykinin peptide binding domaincomprises SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO:191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 or SEQ ID NO: 199.

In other aspects of this embodiment, a tachykinin peptide binding domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO:196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199, at least 75%amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190,SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ IDNO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO:199, at least 80% amino acid identity with SEQ ID NO: 188, SEQ ID NO:189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO:198 OR SEQ ID NO: 199, at least 85% amino acid identity with SEQ ID NO:188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO:197, SEQ ID NO: 198 OR SEQ ID NO: 199, at least 90% amino acid identitywith SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO:196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199 or at least 95%amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190,SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ IDNO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO:199. In yet other aspects of this embodiment, a tachykinin peptidebinding domain comprises a polypeptide having, e.g., at most 70% aminoacid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO:195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199,at most 75% amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO:194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 ORSEQ ID NO: 199, at most 80% amino acid identity with SEQ ID NO: 188, SEQID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO:193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQID NO: 198 OR SEQ ID NO: 199, at most 85% amino acid identity with SEQID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO:192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199, at most 90% amino acididentity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO:191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199 or at most95% amino acid identity with SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO:190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO:199.

In other aspects of this embodiment, a tachykinin peptide binding domaincomprises a polypeptide having, e.g., at least one, two, three, four orfive non-contiguous amino acid substitutions relative to SEQ ID NO: 188,SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ IDNO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197,SEQ ID NO: 198 OR SEQ ID NO: 199. In other aspects of this embodiment, atachykinin peptide binding domain comprises a polypeptide having, e.g.,at most one, two, three, four or five non-contiguous amino acidsubstitutions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO:190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO:199. In yet other aspects of this embodiment, a tachykinin peptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four or five non-contiguous amino acid deletions relative to SEQID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO:192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199. In yet other aspects ofthis embodiment, a tachykinin peptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four or fivenon-contiguous amino acid deletions relative to SEQ ID NO: 188, SEQ IDNO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193,SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ IDNO: 198 OR SEQ ID NO: 199. In still other aspects of this embodiment, atachykinin peptide binding domain comprises a polypeptide having, e.g.,at least one, two, three, four or five non-contiguous amino acidadditions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190,SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ IDNO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO:199. In yet other aspects of this embodiment, a tachykinin peptidebinding domain comprises a polypeptide having, e.g., at most one, two,three, four or five non-contiguous amino acid additions relative to SEQID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO:192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQID NO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199.

In other aspects of this embodiment, a tachykinin peptide binding domaincomprises a polypeptide having, e.g., at least one, two, three, four orfive contiguous amino acid substitutions relative to SEQ ID NO: 188, SEQID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO:193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQID NO: 198 OR SEQ ID NO: 199. In other aspects of this embodiment, atachykinin peptide binding domain comprises a polypeptide having, e.g.,at most one, two, three, four or five contiguous amino acidsubstitutions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO:190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO:199. In yet other aspects of this embodiment, a tachykinin peptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four or five contiguous amino acid deletions relative to SEQ IDNO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192,SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ IDNO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199. In yet other aspects of thisembodiment, a tachykinin peptide binding domain comprises a polypeptidehaving, e.g., at most one, two, three, four or five contiguous aminoacid deletions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO:190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO:199. In still other aspects of this embodiment, a tachykinin peptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four or five contiguous amino acid additions relative to SEQ IDNO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192,SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ IDNO: 197, SEQ ID NO: 198 OR SEQ ID NO: 199. In yet other aspects of thisembodiment, a tachykinin peptide binding domain comprises a polypeptidehaving, e.g., at most one, two, three, four or five contiguous aminoacid additions relative to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO:190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198 OR SEQ ID NO:199.

Another example of a binding domain disclosed in the presentspecification is, e.g., a cholecystokinin peptide, such as, e.g., acholecystokinin 58, a cholecystokinin 39, a cholecystokinin 33, acholecystokinin 12 or a cholecystokinin 8. Thus, in an embodiment, abinding domain comprises a cholecystokinin peptide. In anotherembodiment, a binding domain is derived from a cholecystokinin peptide.

In aspects of this embodiment, a cholecystokinin peptide binding domaincomprises a cholecystokinin 58, a cholecystokinin 39, a cholecystokinin33, a cholecystokinin 12 or a cholecystokinin 8. In other aspects ofthis embodiment, a cholecystokinin peptide comprising a binding domainis SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO:208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In still other aspects ofthis embodiment, a cholecystokinin peptide binding domain comprisesamino acids 20-58 of SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO:208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In yet other aspects ofthis embodiment, a cholecystokinin peptide binding domain comprisesamino acids 26-58 of SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO:208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In still further otheraspects of this embodiment, a cholecystokinin peptide binding domaincomprises amino acids 47-58 of SEQ ID NO: 200, SEQ ID NO: 210 or SEQ IDNO: 214. In yet further aspects of this embodiment, a cholecystokininpeptide binding domain comprises amino acids 51-58 of SEQ ID NO: 200.

In other aspects of this embodiment, a cholecystokinin peptide bindingdomain comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO:203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO:212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215, at least 75%amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202,SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ IDNO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211,SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215, atleast 80% amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO:206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO:215, at least 85% amino acid identity with SEQ ID NO: 200, SEQ ID NO:201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO:210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 orSEQ ID NO: 215, at least 90% amino acid identity with SEQ ID NO: 200,SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ IDNO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209,SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ IDNO: 214 or SEQ ID NO: 215 or at least 95% amino acid identity with SEQID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO:204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO:213, SEQ ID NO: 214 or SEQ ID NO: 215. In yet other aspects of thisembodiment, a cholecystokinin peptide binding domain comprises apolypeptide having, e.g., at most 70% amino acid identity with SEQ IDNO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204,SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ IDNO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213,SEQ ID NO: 214 or SEQ ID NO: 215, at most 75% amino acid identity withSEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ IDNO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208,SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ IDNO: 213, SEQ ID NO: 214 or SEQ ID NO: 215, at most 80% amino acididentity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO:203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO:212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215, at most 85% aminoacid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO:207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215, at most90% amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO:202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO:211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215 orat most 95% amino acid identity with SEQ ID NO: 200, SEQ ID NO: 201, SEQID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO:206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO:215.

In other aspects of this embodiment, a cholecystokinin peptide bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or ten non-contiguous amino acidsubstitutions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO:202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO:211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215.In other aspects of this embodiment, a cholecystokinin peptide bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten non-contiguous amino acidsubstitutions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO:202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO:211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215.In yet other aspects of this embodiment, a cholecystokinin peptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid deletions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO:202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO:211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215.In yet other aspects of this embodiment, a cholecystokinin peptidebinding domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid deletions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO:202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO:211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215.In still other aspects of this embodiment, a cholecystokinin peptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid additions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO:202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO:211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215.In yet other aspects of this embodiment, a cholecystokinin peptidebinding domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid additions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO:202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO:211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215.

In other aspects of this embodiment, a cholecystokinin peptide bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or ten contiguous amino acidsubstitutions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO:202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO:211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215.In other aspects of this embodiment, a cholecystokinin peptide bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten contiguous amino acidsubstitutions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO:202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO:211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215.In yet other aspects of this embodiment, a cholecystokinin peptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or ten contiguous amino aciddeletions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202,SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ IDNO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211,SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In yetother aspects of this embodiment, a cholecystokinin peptide bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten contiguous amino aciddeletions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202,SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ IDNO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211,SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. Instill other aspects of this embodiment, a cholecystokinin peptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202,SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ IDNO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211,SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215. In yetother aspects of this embodiment, a cholecystokinin peptide bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202,SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ IDNO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211,SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215.

In still other aspects of this embodiment, a cholecystokinin peptidebinding domain comprises a polypeptide having, e.g., at least 70% aminoacid identity with amino acids 20-58 of SEQ ID NO: 200 or amino acids26-58 of SEQ ID NO: 200, at least 75% amino acid identity with aminoacids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200, atleast 80% amino acid identity with amino acids 20-58 of SEQ ID NO: 200or amino acids 26-58 of SEQ ID NO: 200, at least 85% amino acid identitywith amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ IDNO: 200, at least 90% amino acid identity with amino acids 20-58 of SEQID NO: 200 or amino acids 26-58 of SEQ ID NO: 200 or at least 95% aminoacid identity with amino acids 20-58 of SEQ ID NO: 200 or amino acids26-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, acholecystokinin peptide binding domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 20-58 of SEQ IDNO: 200 or amino acids 26-58 of SEQ ID NO: 200, at most 75% amino acididentity with amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58of SEQ ID NO: 200, at most 80% amino acid identity with amino acids20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200, at most85% amino acid identity with amino acids 20-58 of SEQ ID NO: 200 oramino acids 26-58 of SEQ ID NO: 200, at most 90% amino acid identitywith amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 of SEQ IDNO: 200 or at most 95% amino acid identity with amino acids 20-58 of SEQID NO: 200 or amino acids 26-58 of SEQ ID NO: 200.

In still other aspects of this embodiment, a cholecystokinin peptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid substitutions relative to amino acids 20-58 of SEQ ID NO: 200 oramino acids 26-58 of SEQ ID NO: 200. In other aspects of thisembodiment, a cholecystokinin peptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid substitutionsrelative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 ofSEQ ID NO: 200. In yet other aspects of this embodiment, acholecystokinin peptide binding domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine orten non-contiguous amino acid deletions relative to amino acids 20-58 ofSEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200. In yet otheraspects of this embodiment, a cholecystokinin peptide binding domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or ten non-contiguous amino acid deletionsrelative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 ofSEQ ID NO: 200. In still other aspects of this embodiment, acholecystokinin peptide binding domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine orten non-contiguous amino acid additions relative to amino acids 20-58 ofSEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200. In yet otheraspects of this embodiment, a cholecystokinin peptide binding domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or ten non-contiguous amino acid additionsrelative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 ofSEQ ID NO: 200.

In still other aspects of this embodiment, a cholecystokinin peptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or ten contiguous amino acidsubstitutions relative to amino acids 20-58 of SEQ ID NO: 200 or aminoacids 26-58 of SEQ ID NO: 200. In other aspects of this embodiment, acholecystokinin peptide binding domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine orten contiguous amino acid substitutions relative to amino acids 20-58 ofSEQ ID NO: 200 or amino acids 26-58 of SEQ ID NO: 200. In yet otheraspects of this embodiment, a cholecystokinin peptide binding domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten contiguous amino acid deletionsrelative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 ofSEQ ID NO: 200. In yet other aspects of this embodiment, acholecystokinin peptide binding domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine orten contiguous amino acid deletions relative to amino acids 20-58 of SEQID NO: 200 or amino acids 26-58 of SEQ ID NO: 200. In still otheraspects of this embodiment, a cholecystokinin peptide binding domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten contiguous amino acid additionsrelative to amino acids 20-58 of SEQ ID NO: 200 or amino acids 26-58 ofSEQ ID NO: 200. In yet other aspects of this embodiment, acholecystokinin peptide binding domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine orten contiguous amino acid additions relative to amino acids 20-58 of SEQID NO: 200 or amino acids 26-58 of SEQ ID NO: 200.

In other aspects of this embodiment, a cholecystokinin peptide bindingdomain comprises a polypeptide having, e.g., at least 70% amino acididentity with amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58of SEQ ID NO: 200, at least 75% amino acid identity with amino acids47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200, at least80% amino acid identity with amino acids 47-58 of SEQ ID NO: 200 oramino acids 51-58 of SEQ ID NO: 200, at least 85% amino acid identitywith amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ IDNO: 200, at least 90% amino acid identity with amino acids 47-58 of SEQID NO: 200 or amino acids 51-58 of SEQ ID NO: 200 or at least 95% aminoacid identity with amino acids 47-58 of SEQ ID NO: 200 or amino acids51-58 of SEQ ID NO: 200. In yet other aspects of this embodiment, acholecystokinin peptide binding domain comprises a polypeptide having,e.g., at most 70% amino acid identity with amino acids 47-58 of SEQ IDNO: 200 or amino acids 51-58 of SEQ ID NO: 200, at most 75% amino acididentity with amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58of SEQ ID NO: 200, at most 80% amino acid identity with amino acids47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200, at most85% amino acid identity with amino acids 47-58 of SEQ ID NO: 200 oramino acids 51-58 of SEQ ID NO: 200, at most 90% amino acid identitywith amino acids 47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ IDNO: 200 or at most 95% amino acid identity with amino acids 47-58 of SEQID NO: 200 or amino acids 51-58 of SEQ ID NO: 200.

In other aspects of this embodiment, a cholecystokinin peptide bindingdomain comprises a polypeptide having, e.g., at least one, two, three orfour non-contiguous amino acid substitutions relative to amino acids47-58 of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In otheraspects of this embodiment, a cholecystokinin peptide binding domaincomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 47-58 ofSEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In yet otheraspects of this embodiment, a cholecystokinin peptide binding domaincomprises a polypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid deletions relative to amino acids 47-58 of SEQID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In yet other aspectsof this embodiment, a cholecystokinin peptide binding domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid deletions relative to amino acids 47-58 of SEQ ID NO: 200 oramino acids 51-58 of SEQ ID NO: 200. In still other aspects of thisembodiment, a cholecystokinin peptide binding domain comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid additions relative to amino acids 47-58 of SEQID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In yet other aspectsof this embodiment, a cholecystokinin peptide binding domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid additions relative to amino acids 47-58 of SEQ ID NO: 200 oramino acids 51-58 of SEQ ID NO: 200.

In other aspects of this embodiment, a cholecystokinin peptide bindingdomain comprises a polypeptide having, e.g., at least one, two, three orfour contiguous amino acid substitutions relative to amino acids 47-58of SEQ ID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In otheraspects of this embodiment, a cholecystokinin peptide binding domaincomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 47-58 of SEQID NO: 200 or amino acids 51-58 of SEQ ID NO: 200. In yet other aspectsof this embodiment, a cholecystokinin peptide binding domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid deletions relative to amino acids 47-58 of SEQ ID NO: 200 oramino acids 51-58 of SEQ ID NO: 200. In yet other aspects of thisembodiment, a cholecystokinin peptide binding domain comprises apolypeptide having, e.g., at most one, two, three or four contiguousamino acid deletions relative to amino acids 47-58 of SEQ ID NO: 200 oramino acids 51-58 of SEQ ID NO: 200. In still other aspects of thisembodiment, a cholecystokinin peptide binding domain comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid additions relative to amino acids 47-58 of SEQ ID NO: 200 oramino acids 51-58 of SEQ ID NO: 200. In yet other aspects of thisembodiment, a cholecystokinin peptide binding domain comprises apolypeptide having, e.g., at most one, two, three or four contiguousamino acid additions relative to amino acids 47-58 of SEQ ID NO: 200 oramino acids 51-58 of SEQ ID NO: 200.

Another example of a binding domain disclosed in the presentspecification is, e.g., a Neuropeptide Y related peptide, such as, e.g.,a Neuropeptide Y (NPY), a Peptide YY (PYY), Pancreatic peptide (PP) or aPancreatic icosapeptide (PIP). Thus, in an embodiment, a binding domaincomprises a Neuropeptide Y related peptide. In another embodiment, abinding domain is derived from a Neuropeptide Y related peptide.

In aspects of this embodiment, a Neuropeptide Y related peptide bindingdomain comprises a Neuropeptide Y (NPY), a Peptide YY (PYY), Pancreaticpeptide (PP) or a Pancreatic icosapeptide (PIP). In other aspects ofthis embodiment, a Neuropeptide Y related peptide binding domaincomprises SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219or SEQ ID NO: 220.

In other aspects of this embodiment, a Neuropeptide Y related peptidebinding domain comprises a polypeptide having, e.g., at least 70% aminoacid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQID NO: 219 or SEQ ID NO: 220, at least 75% amino acid identity with SEQID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO:220, at least 80% amino acid identity with SEQ ID NO: 216, SEQ ID NO:217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220, at least 85%amino acid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218,SEQ ID NO: 219 or SEQ ID NO: 220, at least 90% amino acid identity withSEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ IDNO: 220 or at least 95% amino acid identity with SEQ ID NO: 216, SEQ IDNO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In yet otheraspects of this embodiment, a Neuropeptide Y related peptide bindingdomain comprises a polypeptide having, e.g., at most 70% amino acididentity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO:219 or SEQ ID NO: 220, at most 75% amino acid identity with SEQ ID NO:216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220,at most 80% amino acid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220, at most 85% amino acididentity with SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO:219 or SEQ ID NO: 220, at most 90% amino acid identity with SEQ ID NO:216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220 orat most 95% amino acid identity with SEQ ID NO: 216, SEQ ID NO: 217, SEQID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220.

In other aspects of this embodiment, a Neuropeptide Y related peptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid substitutions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ IDNO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In other aspects of thisembodiment, a Neuropeptide Y related peptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid substitutionsrelative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO:219 or SEQ ID NO: 220. In yet other aspects of this embodiment, aNeuropeptide Y related peptide binding domain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine or ten non-contiguous amino acid deletions relative to SEQ ID NO:216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220.In yet other aspects of this embodiment, a Neuropeptide Y relatedpeptide binding domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid deletions relative to SEQ ID NO: 216, SEQ IDNO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In stillother aspects of this embodiment, a Neuropeptide Y related peptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid additions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219 or SEQ ID NO: 220. In yet other aspects of thisembodiment, a Neuropeptide Y related peptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid additions relativeto SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQID NO: 220.

In other aspects of this embodiment, a Neuropeptide Y related peptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four, five, six, seven, eight, nine or ten contiguous amino acidsubstitutions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219 or SEQ ID NO: 220. In other aspects of thisembodiment, a Neuropeptide Y related peptide binding domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or ten contiguous amino acid substitutions relativeto SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQID NO: 220. In yet other aspects of this embodiment, a Neuropeptide Yrelated peptide binding domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine or tencontiguous amino acid deletions relative to SEQ ID NO: 216, SEQ ID NO:217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220. In yet otheraspects of this embodiment, a Neuropeptide Y related peptide bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or ten contiguous amino aciddeletions relative to SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218,SEQ ID NO: 219 or SEQ ID NO: 220. In still other aspects of thisembodiment, a Neuropeptide Y related peptide binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or ten contiguous amino acid additions relative toSEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ IDNO: 220. In yet other aspects of this embodiment, a Neuropeptide Yrelated peptide binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine or tencontiguous amino acid additions relative to SEQ ID NO: 216, SEQ ID NO:217, SEQ ID NO: 218, SEQ ID NO: 219 or SEQ ID NO: 220.

Another example of a binding domain disclosed in the presentspecification is, e.g., a corticotropin-releasing hormone, athyrotropin-releasing hormone, somatostatin, a leukemia inhibitor factor(LIF) or an interleukin-1 (IL1). Thus, in an embodiment, a bindingdomain comprises a corticotropin-releasing hormone. In an embodiment, abinding domain is derived from a corticotropin-releasing hormone. Inanother embodiment, a binding domain comprises a thyrotropin-releasinghormone. In another embodiment, a binding domain is derived from athyrotropin-releasing hormone. In another embodiment, a binding domaincomprises a somatostatin. In another embodiment, a binding domain isderived from a somatostatin. In another embodiment, a binding domaincomprises a LIF. In another embodiment, a binding domain is derived froma LIF. In another embodiment, a binding domain comprises an IL1. Inanother embodiment, a binding domain is derived from an IL1. In aspectsof this embodiment, a binding domain comprises SEQ ID NO: 221, SEQ IDNO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO:226.

In other aspects of this embodiment, a binding domain comprises apolypeptide having, e.g., at least 70% amino acid identity with SEQ IDNO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225,or SEQ ID NO: 226, at least 75% amino acid identity with SEQ ID NO: 221,SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ IDNO: 226, at least 80% amino acid identity with SEQ ID NO: 221, SEQ IDNO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO:226, at least 85% amino acid identity with SEQ ID NO: 221, SEQ ID NO:222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226,at least 90% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222,SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226 or atleast 95% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226. In yetother aspects of this embodiment, a binding domain comprises apolypeptide having, e.g., at most 70% amino acid identity with SEQ IDNO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225or SEQ ID NO: 226, at most 75% amino acid identity with SEQ ID NO: 221,SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ IDNO: 226, at most 80% amino acid identity with SEQ ID NO: 221, SEQ ID NO:222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226,at most 85% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226, at most90% amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO:223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226 or at most 95%amino acid identity with SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223,SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226.

In other aspects of this embodiment, a binding domain comprises apolypeptide having, e.g., at least one, two, three, four or fivenon-contiguous amino acid substitutions relative to SEQ ID NO: 221, SEQID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO:226. In other aspects of this embodiment, a binding domain comprises apolypeptide having, e.g., at most one, two, three, four or fivenon-contiguous amino acid substitutions relative to SEQ ID NO: 221, SEQID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO:226. In yet other aspects of this embodiment, a binding domain comprisesa polypeptide having, e.g., at least one, two, three, four or fivenon-contiguous amino acid deletions relative to SEQ ID NO: 221, SEQ IDNO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO:226. In yet other aspects of this embodiment, a binding domain comprisesa polypeptide having, e.g., at most one, two, three, four or fivenon-contiguous amino acid deletions relative to SEQ ID NO: 221, SEQ IDNO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO:226. In still other aspects of this embodiment, a binding domaincomprises a polypeptide having, e.g., at least one, two, three, four orfive non-contiguous amino acid additions relative to SEQ ID NO: 221, SEQID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO:226. In yet other aspects of this embodiment, a binding domain comprisesa polypeptide having, e.g., at most one, two, three, four or fivenon-contiguous amino acid additions relative to SEQ ID NO: 221, SEQ IDNO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO:226.

In other aspects of this embodiment, a binding domain comprises apolypeptide having, e.g., at least one, two, three, four or fivecontiguous amino acid substitutions relative to SEQ ID NO: 221, SEQ IDNO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO:226. In other aspects of this embodiment, a binding domain comprises apolypeptide having, e.g., at most one, two, three, four or fivecontiguous amino acid substitutions relative to SEQ ID NO: 221, SEQ IDNO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO:226. In yet other aspects of this embodiment, a binding domain comprisesa polypeptide having, e.g., at least one, two, three, four or fivecontiguous amino acid deletions relative to SEQ ID NO: 221, SEQ ID NO:222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226.In yet other aspects of this embodiment, a binding domain comprises apolypeptide having, e.g., at most one, two, three, four or fivecontiguous amino acid deletions relative to SEQ ID NO: 221, SEQ ID NO:222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226.In still other aspects of this embodiment, a binding domain comprises apolypeptide having, e.g., at least one, two, three, four or fivecontiguous amino acid additions relative to SEQ ID NO: 221, SEQ ID NO:222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226.In yet other aspects of this embodiment, a binding domain comprises apolypeptide having, e.g., at most one, two, three, four or fivecontiguous amino acid additions relative to SEQ ID NO: 221, SEQ ID NO:222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225 or SEQ ID NO: 226.

Another example of a binding domain disclosed in the presentspecification is a kinin peptide, such as, e.g., a bradykinin, akallidin, a desArg⁹ bradykinin and a desArg¹⁰ bradykinin. Thus, in anembodiment, a binding domain comprises a kinin peptide. In anotherembodiment, a binding domain is derived from a kinin peptide. In aspectsof this embodiment, a kinin peptide binding domain comprises abradykinin, a kallidin, a desArg⁹ bradykinin and a desArg¹⁰ bradykinin.In other aspects of this embodiment, a kinin peptide binding domaincomprises SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO:230.

In other aspects of this embodiment, a kinin peptide binding domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230,at least 75% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228,SEQ ID NO: 229 or SEQ ID NO: 230, at least 80% amino acid identity withSEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230, atleast 85% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQID NO: 229 or SEQ ID NO: 230, at least 90% amino acid identity with SEQID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230 or at least95% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO:229 or SEQ ID NO: 230. In yet other aspects of this embodiment, a kininpeptide binding domain comprises a polypeptide having, e.g., at most 70%amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229or SEQ ID NO: 230, at most 75% amino acid identity with SEQ ID NO: 227,SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230, at most 80% amino acididentity with SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ IDNO: 230, at most 85% amino acid identity with SEQ ID NO: 227, SEQ ID NO:228, SEQ ID NO: 229 or SEQ ID NO: 230, at most 90% amino acid identitywith SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230 orat most 95% amino acid identity with SEQ ID NO: 227, SEQ ID NO: 228, SEQID NO: 229 or SEQ ID NO: 230.

In other aspects of this embodiment, a kinin peptide binding domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten non-contiguous amino acidsubstitutions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229or SEQ ID NO: 230. In other aspects of this embodiment, a kinin peptidebinding domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine or ten non-contiguous aminoacid substitutions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ IDNO: 229 or SEQ ID NO: 230. In yet other aspects of this embodiment, akinin peptide binding domain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid deletions relative to SEQ ID NO: 227, SEQ IDNO: 228, SEQ ID NO: 229 or SEQ ID NO: 230. In yet other aspects of thisembodiment, a kinin peptide binding domain comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine or ten non-contiguous amino acid deletions relative to SEQ ID NO:227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230. In still otheraspects of this embodiment, a kinin peptide binding domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid additions relativeto SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230. Inyet other aspects of this embodiment, a kinin peptide binding domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or ten non-contiguous amino acid additionsrelative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO:230.

In other aspects of this embodiment, a kinin peptide binding domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten contiguous amino acid substitutionsrelative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO:230. In other aspects of this embodiment, a kinin peptide binding domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or ten contiguous amino acid substitutionsrelative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO:230. In yet other aspects of this embodiment, a kinin peptide bindingdomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or ten contiguous amino aciddeletions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 orSEQ ID NO: 230. In yet other aspects of this embodiment, a kinin peptidebinding domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine or ten contiguous amino aciddeletions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 orSEQ ID NO: 230. In still other aspects of this embodiment, a kininpeptide binding domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or ten contiguousamino acid additions relative to SEQ ID NO: 227, SEQ ID NO: 228, SEQ IDNO: 229 or SEQ ID NO: 230. In yet other aspects of this embodiment, akinin peptide binding domain comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine or tencontiguous amino acid additions relative to SEQ ID NO: 227, SEQ ID NO:228, SEQ ID NO: 229 or SEQ ID NO: 230.

Another example of a binding domain disclosed in the presentspecification is a PAR peptide, such as, e.g., a PAR1 peptide, a PAR2peptide, a PAR3 peptide and a PAR4 peptide. Thus, in an embodiment, abinding domain comprises a PAR peptide. In another embodiment, a bindingdomain is derived from a PAR peptide. In aspects of this embodiment, aPAR peptide binding domain comprises a PAR1 peptide, a PAR2 peptide, aPAR3 peptide or a PAR4 peptide. In other aspects of this embodiment, aPAR peptide binding domain comprises amino acids 42-47, amino acids42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; aminoacids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 ofSEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234.

In other aspects of this embodiment, a PAR peptide binding domaincomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 42-47, amino acids 42-55, amino acids 29-64 or aminoacids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48,amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 oramino acids 1-70 of SEQ ID NO: 234, at least 75% amino acid identitywith amino acids 42-47, amino acids 42-55, amino acids 29-64 or aminoacids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48,amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 oramino acids 1-70 of SEQ ID NO: 234, at least 80% amino acid identitywith amino acids 42-47, amino acids 42-55, amino acids 29-64 or aminoacids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48,amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 oramino acids 1-70 of SEQ ID NO: 234, at least 85% amino acid identitywith amino acids 42-47, amino acids 42-55, amino acids 29-64 or aminoacids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48,amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 oramino acids 1-70 of SEQ ID NO: 234, at least 90% amino acid identitywith amino acids 42-47, amino acids 42-55, amino acids 29-64 or aminoacids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48,amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 oramino acids 1-70 of SEQ ID NO: 234 or at least 95% amino acid identitywith amino acids 42-47, amino acids 42-55, amino acids 29-64 or aminoacids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48,amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 oramino acids 1-70 of SEQ ID NO: 234.

In yet other aspects of this embodiment, a PAR peptide binding domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 42-47, amino acids 42-55, amino acids 29-64 or aminoacids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48,amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70 oramino acids 1-70 of SEQ ID NO: 234, at most 75% amino acid identity withamino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, aminoacids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44,amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO:233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or aminoacids 1-70 of SEQ ID NO: 234, at most 80% amino acid identity with aminoacids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 ofSEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; aminoacids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 ofSEQ ID NO: 234, at most 85% amino acid identity with amino acids 42-47,amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO:231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or aminoacids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52,amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQID NO: 234, at most 90% amino acid identity with amino acids 42-47,amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO:231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or aminoacids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52,amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQID NO: 234 or at most 95% amino acid identity with amino acids 42-47,amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO:231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or aminoacids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52,amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQID NO: 234.

In other aspects of this embodiment, a PAR peptide binding domaincomprises a polypeptide having, e.g., at least one, two, three, four orfive non-contiguous amino acid substitutions relative to amino acids42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 oramino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; aminoacids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 ofSEQ ID NO: 234. In other aspects of this embodiment, a PAR peptidebinding domain comprises a polypeptide having, e.g., at most one, two,three, four or five non-contiguous amino acid substitutions relative toamino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids 35-48, aminoacids 24-59 or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44,amino acids 39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO:233; amino acids 48-53, amino acids 48-61, amino acids 35-70 or aminoacids 1-70 of SEQ ID NO: 234. In yet other aspects of this embodiment, aPAR peptide binding domain comprises a polypeptide having, e.g., atleast one, two, three, four or five non-contiguous amino acid deletionsrelative to amino acids 42-47, amino acids 42-55, amino acids 29-64 oramino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, amino acids35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232; aminoacids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1-60 ofSEQ ID NO: 233; amino acids 48-53, amino acids 48-61, amino acids 35-70or amino acids 1-70 of SEQ ID NO: 234. In yet other aspects of thisembodiment, a PAR peptide binding domain comprises a polypeptide having,e.g., at most one, two, three, four or five non-contiguous amino aciddeletions relative to amino acids 42-47, amino acids 42-55, amino acids29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40, aminoacids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 232;amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids 48-61, aminoacids 35-70 or amino acids 1-70 of SEQ ID NO: 234. In still otheraspects of this embodiment, a PAR peptide binding domain comprises apolypeptide having, e.g., at least one, two, three, four or fivenon-contiguous amino acid additions relative to amino acids 42-47, aminoacids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231;amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52, aminoacids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53,amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO:234. In yet other aspects of this embodiment, a PAR peptide bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four or five non-contiguous amino acid additions relative to amino acids42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 oramino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; aminoacids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 ofSEQ ID NO: 234.

In other aspects of this embodiment, a PAR peptide binding domaincomprises a polypeptide having, e.g., at least one, two, three, four orfive contiguous amino acid substitutions relative to amino acids 42-47,amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO:231; amino acids 35-40, amino acids 35-48, amino acids 24-59 or aminoacids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids 39-52,amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; amino acids48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 of SEQID NO: 234. In other aspects of this embodiment, a PAR peptide bindingdomain comprises a polypeptide having, e.g., at most one, two, three,four or five contiguous amino acid substitutions relative to amino acids42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 oramino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; aminoacids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 ofSEQ ID NO: 234. In yet other aspects of this embodiment, a PAR peptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four or five contiguous amino acid deletions relative to aminoacids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 ofSEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; aminoacids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 ofSEQ ID NO: 234. In yet other aspects of this embodiment, a PAR peptidebinding domain comprises a polypeptide having, e.g., at most one, two,three, four or five contiguous amino acid deletions relative to aminoacids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 ofSEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; aminoacids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 ofSEQ ID NO: 234. In still other aspects of this embodiment, a PAR peptidebinding domain comprises a polypeptide having, e.g., at least one, two,three, four or five contiguous amino acid additions relative to aminoacids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 ofSEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; aminoacids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 ofSEQ ID NO: 234. In yet other aspects of this embodiment, a PAR peptidebinding domain comprises a polypeptide having, e.g., at most one, two,three, four or five contiguous amino acid additions relative to aminoacids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 ofSEQ ID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59or amino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; aminoacids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 ofSEQ ID NO: 234.

Another example of a binding domain, includes, without limitation, atranslocator, such as, e.g., a protein translocation domain (PTD), like,a herpes simplex virus type 1 VP22 protein translocating sequence, aSV-40 virus large T translocating sequence, a TAT translocatingsequence, an adenovirus translocating sequence, a synthetic integrinbinding domain translocating sequence, a Kaposi fibroblast growth factormembrane translocating sequence, a nuclear localization signal, aTransportan translocating sequence, a ciliary neurotrophic factortranslocating sequence, a caveolin, an interleukin 1-β translocatingsequence, a thioredoxin translocating sequence, a fibroblast growthfactor-1 translocating sequence, a fibroblast growth factor-2translocating sequence, an integrin β1 translocating sequence, anintegrin β3 translocating sequence, a lactoferrin translocatingsequence, a homeodomain translocating sequence, like, a penetratintranslocating sequence, an Engrailed-1 translocating sequence, anEngrailed-2 translocating sequence, a Hoxa-5 translocating sequence, aHoxb-4 translocating sequence, a Hoxc-8 translocating sequence.

Without wishing to limit the invention to any theory or mechanism ofoperation, it is believed that the translocator comprises a proteintranslocation domain (PTD). Further, it is believed that the PTD isprimarily responsible for the translocation of the neurotoxin across acell membrane. PTDs are amino acid sequence domains that have been shownto cross biological membranes efficiently and independently oftransporters or specific receptors. See e.g., Morris M. C. et al.,NATURE BIOTECHNOLOGY, 19:1173-1176 (December 2001), the disclosure ofwhich is herein incorporated by reference in its entirety.

For example, herpes simplex virus type 1 viral protein 22 (HSV-1 VP 22)protein is a transcription factor that concentrates in the nucleus andbinds chromatin. It has been shown that HSV-1 VP 22 comprises atranslocating sequence that mediates trafficking across the membrane vianon-classical endocytosis and can enter cells regardless of GAPjunctions and physical contacts. If HSV-1 VP 22 is expressed in a smallpopulation of cells in culture, it will reach 100% of the cells in thatculture. Fusion proteins with HSV-1 VP 22 and for example p53, GFP,thymidine kinase, β-galactosidase and others have been generated. It hasbeen demonstrated that the fusion proteins are taken up by several kindsof cells including terminally differentiated cells suggesting thatmitosis is not a requirement for efficient entry. In addition, HSV-1 VP22-GFP fusion showed that the protein can shuttle in and out of thecells and enter cells that were not exposed to HSV-1 VP 22.

As another example, the trans-activator gene product (TAT) from thelentivirus HIV-1 was one of the earliest described cell-permeatingproteins comprising a translocating sequence. A receptor-mediated eventappears not to be required for HIV TAT to pass into a neighboring cell;thus for selective cell targeting, a PTD is preferably used inconjunction with a more target cell-selective binding domain in themultivalent Clostridial toxin derivative of the present invention. It isnow known that many other lentiviruses, encodes a potent TAT protein.Embury et al., DIABETES 50:1706-1713, 2001 discuss the use the TAT PTDto transduce anti-apoptotic proteins into pancreatic islet cells. ThePTD of TAT is a small peptide comprising amino acids 47-57 of the TATprotein; the PTD sequence comprises the amino acid sequence YGRKKRRQRRR(SEQ ID NO 41) or at least the nonapeptide comprising TAT amino acids49-57. Fusion proteins comprising this peptide can transit the plasmamembrane in vitro and in vivo. E.g., Schwartz, J. J. et al.,Peptide-mediated cellular delivery, Curr. Opin. Mol. Therapeutics. 2000,2:162-7. The disclosures of these references are incorporated in theirentirety by reference herein.

As another example, a translocating sequence may be derived from ahomeoprotein. Homeoproteins are helix turn helix proteins that contain a60 amino acid DNA-binding domain, the homeodomain. a translocatingsequence may be derived from the homeodomain. One kind of translocatingsequence may be derived from the family of Drosophila homeoproteins.Drosophila homeoproteins are involved in developmental processes and areable to translocate across neuronal membranes. The third helix of thehomeodomain of just 16 amino acids, known as penetratin, is able totranslocate molecules into live cells. When added to several cell typesin culture, 100% of the cells were able to uptake the peptide.Internalization occurs both at 37° C. and 4° C., and thus appears to beneither receptor-mediated nor energy-dependent. Several penetratingpeptides, including those of the penetratin family, have been developedand used to internalize cargo molecules into the cytoplasm and nucleusof several cell types in vivo and in vitro. The results suggest that theentry of penetratin peptides is mediated by tryptophan, phenylalanine,and/or glutamine residues. In addition, the retroinverse and all D-aminoacid forms are also translocated efficiently using the penetratins, andnon-α-helical structures are also internalized. See Prochiantz, A.,Messenger proteins:homeoproteins, TAT and others, Curr Opin Cell Biol2000, 12:400-6; and Schwartz, J J et al., Peptide-mediated cellulardelivery, Curr Opin Mol Therapeutics 2000, 2:162-7. The disclosures ofthese references are incorporated in their entirety by reference herein.

Furthermore, PTD-transported proteins and peptides, such as fusionpeptides, have been shown to generally retain their biologicalproperties and functions once inside the cells. Further, the TAT is ableto carry a variety of cargo molecules including nucleic acid molecules(DNA and RNA), and therapeutic drugs. The capability of this sequence topromote internalization of the peptide (and any cargo peptide) appearsto be related to the strongly positive charge of the PTD sequence, andnot to be inhibited at 4° C. or in the presence of endocytosisinhibitors. Thus, the PTD appears not to be dependent upon activetransport or receptor mediated endocytosis for this activity. The PTDsequence is able to mediate the transduction of its cargo in aconcentration dependent and receptor-, transporter-, andendocytosis-independent manner to 100% of the target cells in a giventissue or culture. Of special interest are the studies demonstratingthat the PTD of TAT is able to deliver proteins in vivo to severaldifferent tissues when injected into animals.

A PTD may comprise a synthetic translocating sequence, such as, e.g.,the synthetic translocating sequence disclosed in, e.g., WO 99/29721 andHo, A. et al., Synthetic PTDs: enhanced transduction potential in vitroand in vivo, CANCER RES 2001, 61, 474-7. In addition, it has beendemonstrated that a 9-mer of L-Arginine is 20 fold more efficient thanthe TAT-PTD at cellular uptake, and when a D-arginine oligomer was usedthe rate enhancement was >100 fold. See Wender, P A et al., The Design,Synthesis, And Evaluation Of Molecules That Enable Or Enhance CellularUptake: Peptoid Molecular Transporters, PROC. NATL. ACAD. SCI. USA 2000,97:13003-13008. These data suggested that the guanidinium groups ofTAT-PTD play a greater role than charge or backbone structure inmediating cellular uptake. Thus, a peptoid analogue containing asix-methylene spacer between the guanidine head group and backbone wassynthesized. This peptoid exhibited enhanced cellular uptake whencompared to TAT-PTD and even to the D-Arg peptide.

In addition to the proteins and peptides discussed above, otherpeptide-mediated delivery systems have been described and are well-knownto those of skill in the art. These PTD-containing proteins and peptidesinclude: MPG, SCWKn, (LARL)n, HA2, RGD, AlkCWK₁₈, DiCWK₁₈, DipaLytic,K₁₆RGD, Plae and Kplae. See Schwartz, J J et al., Peptide-mediatedcellular delivery, Curr Opin Mol Therapeutics 2000, 2:162-7. Thedisclosure of which is incorporated in its entirety by reference herein.In some embodiments, these proteins and peptides may be used astranslocators in accordance with the present invention.

A number of PTDs have now been characterized and are well-known in theart. The amino acid sequences of exemplary translocators are shown inTable 8.

TABLE 8 Translocator Peptides Useful as Binding Domains PTDTranslocating Sequence SEQ ID NO: SV-40 virus large T CGGGPKKKRKVGG 235TAT YGRKKRRQRRR 236 Adenovirus CGGFSTSLRARKA 237 Integrin binding domainCKKKKKKGGRGDMFG 238 Kaposi FGF membrane AAVALLPAVLLALLAP 239translocating sequence Nuclear Localization Signal TPPKKKRKVEDP 240Transportan GWTLNSAGYLLGKINLKALAALAKKIL 241 HSV-1 VP 22DAATATRGRSAASRPTERPRAPARSASRPRRPVE 242 Penetratin 43-58 RQIKIWFQNRRMKWKK243 Penetratin 58-43 KKWKMRRNQFWIKIQR 244 Penetratin 43-58RQIKIWFQNRRMKWKK 245 Penetratin Pro50 RQIKIWFPNRRMKWKK 246 Penetratin3Pro RQPKIWFPNRRMPWKK 247 Penetratin Met-Arg RQIKIWFQNMRRKWKK 248Penetratin 7Arg RQIRIWFQNRRMRWRR 249 Penetratin W/R RRWRRWWRRWWRRWRR 250Penetratin-1 RQIKIFFQNRRMKFKK 251 Penetratin-2 TERQIKIWFQNRRMK 252Penetratin-3 KIWFQNRRMKWKKEN 253

As used herein, a “translocator” of the present invention comprises apolypeptide or a peptidomimetic that facilitates the transport of amolecule across a cell membrane. It is envisioned that both naturallyoccurring translocators as well as non-naturally occurring translocatorscan be used as a binding domain. A translocator includes, withoutlimitation, naturally occurring translocator variants, such as, e.g.,translocator isoforms and translocator subtypes; non-naturally occurringtranslocator variants, such as, e.g., conservative translocatorvariants, non-conservative translocator variants, translocatorchimerics, active translocator fragments thereof, or any combinationthereof.

As used herein, the term “translocator variant,” whethernaturally-occurring or non-naturally-occurring, means a translocatorthat has at least one amino acid change from the corresponding region ofthe disclosed reference sequences (see Table 8) and can be described inpercent identity to the corresponding region of that reference sequence.Unless expressly indicated, all translocator variants disclosed in thepresent specification are capable of executing the cell binding step ofthe intoxication process.

It is recognized by those of skill in the art that there can benaturally occurring translocator variants that differ somewhat in theiramino acid sequence, and also in the nucleic acids encoding theseproteins. For example, HSV-1 VP22 protein variants, TAT variants, andpenetratin variants. As used herein, the term “naturally occurringtranslocator variant” means any translocator produced by anaturally-occurring process, including, without limitation, translocatorisoforms produced from alternatively-spliced transcripts, translocatorisoforms produced by spontaneous mutation and translocator subtypes. Anaturally occurring translocator variant can function in substantiallythe same manner as the reference translocator on which the naturallyoccurring translocator variant is based, and can be substituted for thereference translocator in any aspect of the present invention. Anaturally occurring translocator variant may substitute one or moreamino acids, two or more amino acids, three or more amino acids, four ormore amino acids, five or more amino acids, six or more amino acids,seven or more amino acids, eight or more amino acids, nine or more aminoacids, or ten or more amino acids from the reference translocator onwhich the naturally occurring translocator variant is based. A naturallyoccurring translocator variant can also substitute at least 2 contiguousamino acids, at least 3 contiguous amino acids, at least 4 contiguousamino acids, at least 5 contiguous amino acids, at least 6 contiguousamino acids, at least 7 contiguous amino acids, at least 8 contiguousamino acids, at least 9 contiguous amino acids, or at least 10contiguous amino acids from the reference translocator on which thenaturally occurring translocator variant is based, that possess at least50% amino acid identity, 65% amino acid identity, 75% amino acididentity, 85% amino acid identity or 95% amino acid identity to thereference translocator on which the naturally occurring translocatorvariant is based.

A non-limiting examples of a naturally occurring translocator variant isa translocator isoform such as, e.g., a HSV-1 VP22 protein isoform, aSV-40 virus large T isoform, a TAT isoform, an adenovirus isoform, asynthetic integrin binding domain isoform, a Kaposi fibroblast growthfactor membrane isoform, a nuclear localization signal, a Transportanisoform, a ciliary neurotrophic factor isoform, a caveolin, aninterleukin 1-β isoform, a thioredoxin isoform, a fibroblast growthfactor-1 isoform, a fibroblast growth factor-2 isoform, an integrin β1isoform, an integrin β3 isoform, a lactoferrin isoform, a homeodomainisoform, like, a penetratin isoform, an Engrailed-1 isoform, anEngrailed-2 isoform, a Hoxa-5 isoform, a Hoxb-4 isoform, a Hoxc-8isoform. A translocator isoform can function in substantially the samemanner as the reference translocator on which the translocator isoformis based, and can be substituted for the reference translocator in anyaspect of the present invention.

Another non-limiting examples of a naturally occurring translocatorvariant is a translocator subtype such as, e.g., a HSV-1 VP22 proteinsubtype, a SV-40 virus large T subtype, a TAT subtype, an adenovirussubtype, a synthetic integrin binding domain subtype, a Kaposifibroblast growth factor membrane subtype, a nuclear localizationsignal, a Transportan subtype, a ciliary neurotrophic factor subtype, acaveolin, an interleukin 1-β subtype, a thioredoxin subtype, afibroblast growth factor-1 subtype, a fibroblast growth factor-2subtype, an integrin β1 subtype, an integrin β3 subtype, a lactoferrinsubtype, a homeodomain subtype, like, a penetratin subtype, anEngrailed-1 subtype, an Engrailed-2 subtype, a Hoxa-5 subtype, a Hoxb-4subtype, a Hoxc-8 subtype. A translocator subtype can function insubstantially the same manner as the reference translocator on which thetranslocator subtype is based, and can be substituted for the referencetranslocator in any aspect of the present invention.

As used herein, the term “non-naturally occurring translocator variant”means any translocator produced with the aid of human manipulation,including, without limitation, translocators produced by geneticengineering using random mutagenesis or rational design andtranslocators produced by chemical synthesis. Non-limiting examples ofnon-naturally occurring translocator variants include, e.g.,conservative translocator variants, non-conservative translocatorvariants, translocator chimeric variants and active translocatorfragments.

As used herein, the term “conservative translocator variant” means atranslocator that has at least one amino acid substituted by anotheramino acid or an amino acid analog that has at least one propertysimilar to that of the original amino acid from the referencetranslocator sequence (see Table 8). Examples of properties include,without limitation, similar size, topography, charge, hydrophobicity,hydrophilicity, lipophilicity, covalent-bonding capacity,hydrogen-bonding capacity, a physicochemical property, of the like, orany combination thereof. A conservative translocator variant canfunction in substantially the same manner as the reference translocatoron which the conservative translocator variant is based, and can besubstituted for the reference translocator in any aspect of the presentinvention. A conservative translocator variant may substitute one ormore amino acids, two or more amino acids, three or more amino acids,four or more amino acids, five or more amino acids, six or more aminoacids, seven or more amino acids, eight or more amino acids, nine ormore amino acids, or ten or more amino acids from the referencetranslocator on which the conservative translocator variant is based. Aconservative translocator variant can also substitute at least 2contiguous amino acids, at least 3 contiguous amino acids, at least 4contiguous amino acids, at least 5 contiguous amino acids, at least 6contiguous amino acids, at least 7 contiguous amino acids, at least 8contiguous amino acids, at least 9 contiguous amino acids, or at least10 contiguous amino acids from the reference translocator on which theconservative translocator variant is based, that possess at least 50%amino acid identity, 65% amino acid identity, 75% amino acid identity,85% amino acid identity or 95% amino acid identity to the referencetranslocator on which the conservative translocator variant is based.Non-limiting examples of a conservative translocator variant include,e.g., a conservative HSV-1 VP22 protein variant, a conservative SV-40virus large T variant, a conservative TAT variant, an adenovirusvariant, a conservative synthetic integrin binding domain variant, aconservative Kaposi fibroblast growth factor membrane variant, aconservative nuclear localization signal, a conservative Transportanvariant, a conservative ciliary neurotrophic factor variant, aconservative caveolin, an interleukin 1-β variant, a conservativethioredoxin variant, a conservative fibroblast growth factor-1 variant,a conservative fibroblast growth factor-2 variant, an integrin β1variant, an integrin β3 variant, a conservative lactoferrin variant, aconservative homeodomain variant, like, a conservative penetratinvariant, an Engrailed-1 variant, an Engrailed-2 variant, a conservativeHoxa-5 variant, a conservative Hoxb-4 variant, a conservative Hoxc-8variant.

As used herein, the term “non-conservative translocator variant” means atranslocator in which 1) at least one amino acid is deleted from thereference translocator on which the non-conservative translocatorvariant is based; 2) at least one amino acid added to the referencetranslocator on which the non-conservative translocator is based; or 3)at least one amino acid is substituted by another amino acid or an aminoacid analog that does not share any property similar to that of theoriginal amino acid from the reference translocator sequence (see Table8). A non-conservative translocator variant can function insubstantially the same manner as the reference translocator on which thenon-conservative translocator variant is based, and can be substitutedfor the reference translocator in any aspect of the present invention. Anon-conservative translocator variant can delete one or more aminoacids, two or more amino acids, three or more amino acids, four or moreamino acids, five or more amino acids, and ten or more amino acids fromthe reference translocator on which the non-conservative translocatorvariant is based. A non-conservative translocator variant can add one ormore amino acids, two or more amino acids, three or more amino acids,four or more amino acids, five or more amino acids, and ten or moreamino acids to the reference translocator on which the non-conservativetranslocator variant is based. A non-conservative translocator variantmay substitute one or more amino acids, two or more amino acids, threeor more amino acids, four or more amino acids, five or more amino acids,six or more amino acids, seven or more amino acids, eight or more aminoacids, nine or more amino acids, or ten or more amino acids from thereference translocator on which the non-conservative translocatorvariant is based. A non-conservative translocator variant can alsosubstitute at least 2 contiguous amino acids, at least 3 contiguousamino acids, at least 4 contiguous amino acids, at least 5 contiguousamino acids, at least 6 contiguous amino acids, at least 7 contiguousamino acids, at least 8 contiguous amino acids, at least 9 contiguousamino acids, or at least 10 contiguous amino acids from the referencetranslocator on which the non-conservative translocator variant isbased, that possess at least 50% amino acid identity, 65% amino acididentity, 75% amino acid identity, 85% amino acid identity or 95% aminoacid identity to the reference translocator on which thenon-conservative translocator variant is based. Non-limiting examples ofa non-conservative translocator variant include, e.g., anon-conservative HSV-1 VP22 protein variant, a non-conservative SV-40virus large T variant, a non-conservative TAT variant, an adenovirusvariant, a non-conservative synthetic integrin binding domain variant, anon-conservative Kaposi fibroblast growth factor membrane variant, anon-conservative nuclear localization signal, a non-conservativeTransportan variant, a non-conservative ciliary neurotrophic factorvariant, a non-conservative caveolin, an interleukin 1-β variant, anon-conservative thioredoxin variant, a non-conservative fibroblastgrowth factor-1 variant, a non-conservative fibroblast growth factor-2variant, an integrin β1 variant, an integrin β3 variant, anon-conservative lactoferrin variant, a non-conservative homeodomainvariant, like, a non-conservative penetratin variant, an Engrailed-1variant, an Engrailed-2 variant, a non-conservative Hoxa-5 variant, anon-conservative Hoxb-4 variant, a non-conservative Hoxc-8 variant.

As used herein, the term “translocator chimeric” means a polypeptidecomprising at least a portion of a translocator and at least a portionof at least one other polypeptide to form an enhanced targeting domainwith at least one property different from the reference translocator(see Table 8), with the proviso that this translocator chimeric canfacilitate the transport of a molecule across a cell membrane.

As used herein, the term “active translocator fragment” means any of avariety of translocator fragments comprising the enhanced targetingdomain can be useful in aspects of the present invention with theproviso that these NAP fragments can facilitate the transport of amolecule across a cell membrane.

In some embodiments, the translocator may function independently of cellsurface transporters or specific receptors, and thereforenon-specifically. In this embodiment the multivalent Clostridial toxinwill also comprise at least one target cell selective ligand. In someembodiments, one or more binding domain of the multivalent Clostridialtoxin may comprise a translocator. In other embodiments, a translocatormay comprise a peptide or peptidomimetic selective for a cell surfacetransporter, feature, or receptor; in such an embodiment the multivalentClostridial toxin may comprise two or more identical binding domains, ormay comprise at least one target cell selective domain and at least onetarget cell non-selective binding domain, so long as the number ofbinding domains totals at least two.

In an embodiment, a binding domain comprises a translocator. In aspectsof this embodiment, a translocator comprises a PTD.

In another embodiment, a binding domain comprises a translocatingsequence comprises a SV-40 virus large T translocating sequence. Inanother embodiment, a binding domain comprises a translocating sequencederived from a SV-40 virus large T translocating sequence. In an aspectof this embodiment, a binding domain comprises a SV-40 virus large Ttranslocating sequence comprises SEQ ID NO: 235. In another aspect ofthis embodiment, a binding domain comprises a SV-40 virus large Ttranslocating sequence derived from SEQ ID NO: 235.

In other aspects of this embodiment, a SV-40 virus large T translocatingsequence comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 235, at least 75% amino acid identity with SEQID NO: 235, at least 80% amino acid identity with SEQ ID NO: 235, atleast 85% amino acid identity with SEQ ID NO: 235, at least 90% aminoacid identity with SEQ ID NO: 235 or at least 95% amino acid identitywith SEQ ID NO: 235. In yet other aspects of this embodiment, a SV-40virus large T translocating sequence comprises a polypeptide having,e.g., at most 70% amino acid identity with SEQ ID NO: 235, at most 75%amino acid identity with SEQ ID NO: 235, at most 80% amino acid identitywith SEQ ID NO: 235, at most 85% amino acid identity with SEQ ID NO:235, at most 90% amino acid identity with SEQ ID NO: 235 or at most 95%amino acid identity with SEQ ID NO: 235.

In other aspects of this embodiment, a SV-40 virus large T translocatingsequence comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 235. In other aspects of thisembodiment, a SV-40 virus large T translocating sequence comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, or 10 non-contiguous amino acid substitutionsrelative to SEQ ID NO: 235. In yet other aspects of this embodiment, aSV-40 virus large T translocating sequence comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO:235. In other aspects of this embodiment, a SV-40 virus large Ttranslocating sequence comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid deletions relative to SEQ ID NO: 235. In stillother aspects of this embodiment, a SV-40 virus large T translocatingsequence comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, or 10 non-contiguous amino acidadditions relative to SEQ ID NO: 235. In other aspects of thisembodiment, a SV-40 virus large T translocating sequence comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, or 10 non-contiguous amino acid additions relativeto SEQ ID NO: 235.

In other aspects of this embodiment, a SV-40 virus large T translocatingsequence comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, or 10 contiguous amino acidsubstitutions relative to SEQ ID NO: 235. In other aspects of thisembodiment, a SV-40 virus large T translocating sequence comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, or 10 contiguous amino acid substitutions relativeto SEQ ID NO: 235. In yet other aspects of this embodiment, a SV-40virus large T translocating sequence comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, or10 contiguous amino acid deletions relative to SEQ ID NO: 235. In otheraspects of this embodiment, a SV-40 virus large T translocating sequencecomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, or 10 contiguous amino acid deletionsrelative to SEQ ID NO: 235. In still other aspects of this embodiment, aSV-40 virus large T translocating sequence comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 235.In other aspects of this embodiment, a SV-40 virus large T translocatingsequence comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, or 10 contiguous amino acidadditions relative to SEQ ID NO: 235.

In another embodiment, a binding domain comprises a translocatingsequence comprises a TAT translocating sequence. In another embodiment,a binding domain comprises a translocating sequence derived from a TATtranslocating sequence. In an aspect of this embodiment, a bindingdomain comprises a TAT translocating sequence comprises SEQ ID NO: 236.In another aspect of this embodiment, a binding domain comprises a TATtranslocating sequence derived from SEQ ID NO: 236.

In other aspects of this embodiment, a TAT translocating sequencecomprises a polypeptide having, e.g., at least 70% amino acid identitywith SEQ ID NO: 236, at least 75% amino acid identity with SEQ ID NO:236, at least 80% amino acid identity with SEQ ID NO: 236, at least 85%amino acid identity with SEQ ID NO: 236, at least 90% amino acididentity with SEQ ID NO: 236 or at least 95% amino acid identity withSEQ ID NO: 236. In yet other aspects of this embodiment, a TATtranslocating sequence comprises a polypeptide having, e.g., at most 70%amino acid identity with SEQ ID NO: 236, at most 75% amino acid identitywith SEQ ID NO: 236, at most 80% amino acid identity with SEQ ID NO:236, at most 85% amino acid identity with SEQ ID NO: 236, at most 90%amino acid identity with SEQ ID NO: 236 or at most 95% amino acididentity with SEQ ID NO: 236.

In other aspects of this embodiment, a TAT translocating sequencecomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 236. In other aspects of thisembodiment, a TAT translocating sequence comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, or10 non-contiguous amino acid substitutions relative to SEQ ID NO: 236.In yet other aspects of this embodiment, a TAT translocating sequencecomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, or 10 non-contiguous amino acid deletionsrelative to SEQ ID NO: 236. In other aspects of this embodiment, a TATtranslocating sequence comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid deletions relative to SEQ ID NO: 236. In stillother aspects of this embodiment, a TAT translocating sequence comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, or 10 non-contiguous amino acid additions relativeto SEQ ID NO: 236. In other aspects of this embodiment, a TATtranslocating sequence comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid additions relative to SEQ ID NO: 236.

In other aspects of this embodiment, a TAT translocating sequencecomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, or 10 contiguous amino acid substitutionsrelative to SEQ ID NO: 236. In other aspects of this embodiment, a TATtranslocating sequence comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, or 10 contiguousamino acid substitutions relative to SEQ ID NO: 236. In yet otheraspects of this embodiment, a TAT translocating sequence comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, or 10 contiguous amino acid deletions relative toSEQ ID NO: 236. In other aspects of this embodiment, a TAT translocatingsequence comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, or 10 contiguous amino aciddeletions relative to SEQ ID NO: 236. In still other aspects of thisembodiment, a TAT translocating sequence comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, or10 contiguous amino acid additions relative to SEQ ID NO: 236. In otheraspects of this embodiment, a TAT translocating sequence comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, or 10 contiguous amino acid additions relative toSEQ ID NO: 236.

In another embodiment, a binding domain comprises a translocatingsequence comprises an adenovirus translocating sequence. In anotherembodiment, a binding domain comprises a translocating sequence derivedfrom an adenovirus translocating sequence. In an aspect of thisembodiment, a binding domain comprises an adenovirus translocatingsequence comprises SEQ ID NO: 237. In another aspect of this embodiment,a binding domain comprises an adenovirus translocating sequence derivedfrom SEQ ID NO: 237.

In other aspects of this embodiment, an adenovirus translocatingsequence comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 237, at least 75% amino acid identity with SEQID NO: 237, at least 80% amino acid identity with SEQ ID NO: 237, atleast 85% amino acid identity with SEQ ID NO: 237, at least 90% aminoacid identity with SEQ ID NO: 237 or at least 95% amino acid identitywith SEQ ID NO: 237. In yet other aspects of this embodiment, anadenovirus translocating sequence comprises a polypeptide having, e.g.,at most 70% amino acid identity with SEQ ID NO: 237, at most 75% aminoacid identity with SEQ ID NO: 237, at most 80% amino acid identity withSEQ ID NO: 237, at most 85% amino acid identity with SEQ ID NO: 237, atmost 90% amino acid identity with SEQ ID NO: 237 or at most 95% aminoacid identity with SEQ ID NO: 237.

In other aspects of this embodiment, an adenovirus translocatingsequence comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 237. In other aspects of thisembodiment, an adenovirus translocating sequence comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, or 10 non-contiguous amino acid substitutions relative to SEQ IDNO: 237. In yet other aspects of this embodiment, an adenovirustranslocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid deletions relative to SEQ ID NO: 237. In otheraspects of this embodiment, an adenovirus translocating sequencecomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, or 10 non-contiguous amino acid deletionsrelative to SEQ ID NO: 237. In still other aspects of this embodiment,an adenovirus translocating sequence comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, or10 non-contiguous amino acid additions relative to SEQ ID NO: 237. Inother aspects of this embodiment, an adenovirus translocating sequencecomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, or 10 non-contiguous amino acid additionsrelative to SEQ ID NO: 237.

In other aspects of this embodiment, an adenovirus translocatingsequence comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, or 10 contiguous amino acidsubstitutions relative to SEQ ID NO: 237. In other aspects of thisembodiment, an adenovirus translocating sequence comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO:237. In yet other aspects of this embodiment, an adenovirustranslocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10 contiguousamino acid deletions relative to SEQ ID NO: 237. In other aspects ofthis embodiment, an adenovirus translocating sequence comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, or 10 contiguous amino acid deletions relative toSEQ ID NO: 237. In still other aspects of this embodiment, an adenovirustranslocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10 contiguousamino acid additions relative to SEQ ID NO: 237. In other aspects ofthis embodiment, an adenovirus translocating sequence comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, or 10 contiguous amino acid additions relative toSEQ ID NO: 237.

In another embodiment, a binding domain comprises a translocatingsequence comprises an integrin binding domain translocating sequence. Inanother embodiment, a binding domain comprises a translocating sequencederived from an integrin binding domain translocating sequence. In anaspect of this embodiment, a binding domain comprises an integrinbinding domain translocating sequence comprises SEQ ID NO: 238. Inanother aspect of this embodiment, a binding domain comprises anintegrin binding domain translocating sequence derived from SEQ ID NO:238.

In other aspects of this embodiment, an integrin binding domaintranslocating sequence comprises a polypeptide having, e.g., at least70% amino acid identity with SEQ ID NO: 238, at least 75% amino acididentity with SEQ ID NO: 238, at least 80% amino acid identity with SEQID NO: 238, at least 85% amino acid identity with SEQ ID NO: 238, atleast 90% amino acid identity with SEQ ID NO: 238 or at least 95% aminoacid identity with SEQ ID NO: 238. In yet other aspects of thisembodiment, an integrin binding domain translocating sequence comprisesa polypeptide having, e.g., at most 70% amino acid identity with SEQ IDNO: 238, at most 75% amino acid identity with SEQ ID NO: 238, at most80% amino acid identity with SEQ ID NO: 238, at most 85% amino acididentity with SEQ ID NO: 238, at most 90% amino acid identity with SEQID NO: 238 or at most 95% amino acid identity with SEQ ID NO: 238.

In other aspects of this embodiment, an integrin binding domaintranslocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid substitutions relative to SEQ ID NO: 238. Inother aspects of this embodiment, an integrin binding domaintranslocating sequence comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid substitutions relative to SEQ ID NO: 238. Inyet other aspects of this embodiment, an integrin binding domaintranslocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid deletions relative to SEQ ID NO: 238. In otheraspects of this embodiment, an integrin binding domain translocatingsequence comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, or 10 non-contiguous amino aciddeletions relative to SEQ ID NO: 238. In still other aspects of thisembodiment, an integrin binding domain translocating sequence comprisesa polypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, or 10 non-contiguous amino acid additions relativeto SEQ ID NO: 238. In other aspects of this embodiment, an integrinbinding domain translocating sequence comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, or10 non-contiguous amino acid additions relative to SEQ ID NO: 238.

In other aspects of this embodiment, an integrin binding domaintranslocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10 contiguousamino acid substitutions relative to SEQ ID NO: 238. In other aspects ofthis embodiment, an integrin binding domain translocating sequencecomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, or 10 contiguous amino acid substitutionsrelative to SEQ ID NO: 238. In yet other aspects of this embodiment, anintegrin binding domain translocating sequence comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 238.In other aspects of this embodiment, an integrin binding domaintranslocating sequence comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, or 10 contiguousamino acid deletions relative to SEQ ID NO: 238. In still other aspectsof this embodiment, an integrin binding domain translocating sequencecomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, or 10 contiguous amino acid additionsrelative to SEQ ID NO: 238. In other aspects of this embodiment, anintegrin binding domain translocating sequence comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 238.

In another embodiment, a binding domain comprises a translocatingsequence comprises a Kaposi FGF membrane translocating sequence. Inanother embodiment, a binding domain comprises a translocating sequencederived from a Kaposi FGF membrane translocating sequence. In an aspectof this embodiment, a binding domain comprises a Kaposi FGF membranetranslocating sequence comprises SEQ ID NO: 239. In another aspect ofthis embodiment, a binding domain comprises a Kaposi FGF membranetranslocating sequence derived from SEQ ID NO: 239.

In other aspects of this embodiment, a Kaposi FGF membrane translocatingsequence comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 239, at least 75% amino acid identity with SEQID NO: 239, at least 80% amino acid identity with SEQ ID NO: 239, atleast 85% amino acid identity with SEQ ID NO: 239, at least 90% aminoacid identity with SEQ ID NO: 239 or at least 95% amino acid identitywith SEQ ID NO: 239. In yet other aspects of this embodiment, a KaposiFGF membrane translocating sequence comprises a polypeptide having,e.g., at most 70% amino acid identity with SEQ ID NO: 239, at most 75%amino acid identity with SEQ ID NO: 239, at most 80% amino acid identitywith SEQ ID NO: 239, at most 85% amino acid identity with SEQ ID NO:239, at most 90% amino acid identity with SEQ ID NO: 239 or at most 95%amino acid identity with SEQ ID NO: 239.

In other aspects of this embodiment, a Kaposi FGF membrane translocatingsequence comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 239. In other aspects of thisembodiment, a Kaposi FGF membrane translocating sequence comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, or 10 non-contiguous amino acid substitutionsrelative to SEQ ID NO: 239. In yet other aspects of this embodiment, aKaposi FGF membrane translocating sequence comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, or 10 non-contiguous amino acid deletions relative to SEQ ID NO:239. In other aspects of this embodiment, a Kaposi FGF membranetranslocating sequence comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid deletions relative to SEQ ID NO: 239. In stillother aspects of this embodiment, a Kaposi FGF membrane translocatingsequence comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, or 10 non-contiguous amino acidadditions relative to SEQ ID NO: 239. In other aspects of thisembodiment, a Kaposi FGF membrane translocating sequence comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, or 10 non-contiguous amino acid additions relativeto SEQ ID NO: 239.

In other aspects of this embodiment, a Kaposi FGF membrane translocatingsequence comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, or 10 contiguous amino acidsubstitutions relative to SEQ ID NO: 239. In other aspects of thisembodiment, a Kaposi FGF membrane translocating sequence comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, or 10 contiguous amino acid substitutions relativeto SEQ ID NO: 239. In yet other aspects of this embodiment, a Kaposi FGFmembrane translocating sequence comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, or 10contiguous amino acid deletions relative to SEQ ID NO: 239. In otheraspects of this embodiment, a Kaposi FGF membrane translocating sequencecomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, or 10 contiguous amino acid deletionsrelative to SEQ ID NO: 239. In still other aspects of this embodiment, aKaposi FGF membrane translocating sequence comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, or 10 contiguous amino acid additions relative to SEQ ID NO: 239.In other aspects of this embodiment, a Kaposi FGF membrane translocatingsequence comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, or 10 contiguous amino acidadditions relative to SEQ ID NO: 239.

In another embodiment, a binding domain comprises a translocatingsequence comprises a Nuclear Localization Signal translocating sequence.In another embodiment, a binding domain comprises a translocatingsequence derived from a Nuclear Localization Signal translocatingsequence. In an aspect of this embodiment, a binding domain comprises aNuclear Localization Signal translocating sequence comprises SEQ ID NO:240. In another aspect of this embodiment, a binding domain comprises aNuclear Localization Signal translocating sequence derived from SEQ IDNO: 240.

In other aspects of this embodiment, a Nuclear Localization Signaltranslocating sequence comprises a polypeptide having, e.g., at least70% amino acid identity with SEQ ID NO: 240, at least 75% amino acididentity with SEQ ID NO: 240, at least 80% amino acid identity with SEQID NO: 240, at least 85% amino acid identity with SEQ ID NO: 240, atleast 90% amino acid identity with SEQ ID NO: 240 or at least 95% aminoacid identity with SEQ ID NO: 240. In yet other aspects of thisembodiment, a Nuclear Localization Signal translocating sequencecomprises a polypeptide having, e.g., at most 70% amino acid identitywith SEQ ID NO: 240, at most 75% amino acid identity with SEQ ID NO:240, at most 80% amino acid identity with SEQ ID NO: 240, at most 85%amino acid identity with SEQ ID NO: 240, at most 90% amino acid identitywith SEQ ID NO: 240 or at most 95% amino acid identity with SEQ ID NO:240.

In other aspects of this embodiment, a Nuclear Localization Signaltranslocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid substitutions relative to SEQ ID NO: 240. Inother aspects of this embodiment, a Nuclear Localization Signaltranslocating sequence comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid substitutions relative to SEQ ID NO: 240. Inyet other aspects of this embodiment, a Nuclear Localization Signaltranslocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid deletions relative to SEQ ID NO: 240. In otheraspects of this embodiment, a Nuclear Localization Signal translocatingsequence comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, or 10 non-contiguous amino aciddeletions relative to SEQ ID NO: 240. In still other aspects of thisembodiment, a Nuclear Localization Signal translocating sequencecomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, or 10 non-contiguous amino acid additionsrelative to SEQ ID NO: 240. In other aspects of this embodiment, aNuclear Localization Signal translocating sequence comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, or 10 non-contiguous amino acid additions relativeto SEQ ID NO: 240.

In other aspects of this embodiment, a Nuclear Localization Signaltranslocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10 contiguousamino acid substitutions relative to SEQ ID NO: 240. In other aspects ofthis embodiment, a Nuclear Localization Signal translocating sequencecomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, or 10 contiguous amino acid substitutionsrelative to SEQ ID NO: 240. In yet other aspects of this embodiment, aNuclear Localization Signal translocating sequence comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, or 10 contiguous amino acid deletions relative toSEQ ID NO: 240. In other aspects of this embodiment, a NuclearLocalization Signal translocating sequence comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, or 10 contiguous amino acid deletions relative to SEQ ID NO: 240.In still other aspects of this embodiment, a Nuclear Localization Signaltranslocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10 contiguousamino acid additions relative to SEQ ID NO: 240. In other aspects ofthis embodiment, a Nuclear Localization Signal translocating sequencecomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, or 10 contiguous amino acid additionsrelative to SEQ ID NO: 240.

In another embodiment, a binding domain comprises a translocatingsequence comprises a Transportan translocating sequence. In anotherembodiment, a binding domain comprises a translocating sequence derivedfrom a Transportan translocating sequence. In an aspect of thisembodiment, a binding domain comprises a Transportan translocatingsequence comprises SEQ ID NO: 241. In another aspect of this embodiment,a binding domain comprises a Transportan translocating sequence derivedfrom SEQ ID NO: 241.

In other aspects of this embodiment, a Transportan translocatingsequence comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 241, at least 75% amino acid identity with SEQID NO: 241, at least 80% amino acid identity with SEQ ID NO: 241, atleast 85% amino acid identity with SEQ ID NO: 241, at least 90% aminoacid identity with SEQ ID NO: 241 or at least 95% amino acid identitywith SEQ ID NO: 241. In yet other aspects of this embodiment, aTransportan translocating sequence comprises a polypeptide having, e.g.,at most 70% amino acid identity with SEQ ID NO: 241, at most 75% aminoacid identity with SEQ ID NO: 241, at most 80% amino acid identity withSEQ ID NO: 241, at most 85% amino acid identity with SEQ ID NO: 241, atmost 90% amino acid identity with SEQ ID NO: 241 or at most 95% aminoacid identity with SEQ ID NO: 241.

In other aspects of this embodiment, a Transportan translocatingsequence comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 241. In other aspects of thisembodiment, a Transportan translocating sequence comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, or 10 non-contiguous amino acid substitutions relative to SEQ IDNO: 241. In yet other aspects of this embodiment, a Transportantranslocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid deletions relative to SEQ ID NO: 241. In otheraspects of this embodiment, a Transportan translocating sequencecomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, or 10 non-contiguous amino acid deletionsrelative to SEQ ID NO: 241. In still other aspects of this embodiment, aTransportan translocating sequence comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid additions relative to SEQ ID NO: 241. In otheraspects of this embodiment, a Transportan translocating sequencecomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, or 10 non-contiguous amino acid additionsrelative to SEQ ID NO: 241.

In other aspects of this embodiment, a Transportan translocatingsequence comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, or 10 contiguous amino acidsubstitutions relative to SEQ ID NO: 241. In other aspects of thisembodiment, a Transportan translocating sequence comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO:241. In yet other aspects of this embodiment, a Transportantranslocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10 contiguousamino acid deletions relative to SEQ ID NO: 241. In other aspects ofthis embodiment, a Transportan translocating sequence comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, or 10 contiguous amino acid deletions relative toSEQ ID NO: 241. In still other aspects of this embodiment, a Transportantranslocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10 contiguousamino acid additions relative to SEQ ID NO: 241. In other aspects ofthis embodiment, a Transportan translocating sequence comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, or 10 contiguous amino acid additions relative toSEQ ID NO: 241.

In another embodiment, a binding domain comprises a translocatingsequence comprises a HSV-1 VP 22 translocating sequence. In anotherembodiment, a binding domain comprises a translocating sequence derivedfrom a HSV-1 VP 22 translocating sequence. In an aspect of thisembodiment, a binding domain comprises a HSV-1 VP 22 translocatingsequence comprises SEQ ID NO: 242. In another aspect of this embodiment,a binding domain comprises a HSV-1 VP 22 translocating sequence derivedfrom SEQ ID NO: 242.

In other aspects of this embodiment, a HSV-1 VP 22 translocatingsequence comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 242, at least 75% amino acid identity with SEQID NO: 242, at least 80% amino acid identity with SEQ ID NO: 242, atleast 85% amino acid identity with SEQ ID NO: 242, at least 90% aminoacid identity with SEQ ID NO: 242 or at least 95% amino acid identitywith SEQ ID NO: 242. In yet other aspects of this embodiment, a HSV-1 VP22 translocating sequence comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 242, at most 75% amino acididentity with SEQ ID NO: 242, at most 80% amino acid identity with SEQID NO: 242, at most 85% amino acid identity with SEQ ID NO: 242, at most90% amino acid identity with SEQ ID NO: 242 or at most 95% amino acididentity with SEQ ID NO: 242.

In other aspects of this embodiment, a HSV-1 VP 22 translocatingsequence comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 242. In other aspects of thisembodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, or 10 non-contiguous amino acid substitutions relative to SEQ IDNO: 242. In yet other aspects of this embodiment, a HSV-1 VP 22translocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid deletions relative to SEQ ID NO: 242. In otheraspects of this embodiment, a HSV-1 VP 22 translocating sequencecomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, or 10 non-contiguous amino acid deletionsrelative to SEQ ID NO: 242. In still other aspects of this embodiment, aHSV-1 VP 22 translocating sequence comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid additions relative to SEQ ID NO: 242. In otheraspects of this embodiment, a HSV-1 VP 22 translocating sequencecomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, or 10 non-contiguous amino acid additionsrelative to SEQ ID NO: 242.

In other aspects of this embodiment, a HSV-1 VP 22 translocatingsequence comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, or 10 contiguous amino acidsubstitutions relative to SEQ ID NO: 242. In other aspects of thisembodiment, a HSV-1 VP 22 translocating sequence comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, or 10 contiguous amino acid substitutions relative to SEQ ID NO:242. In yet other aspects of this embodiment, a HSV-1 VP 22translocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10 contiguousamino acid deletions relative to SEQ ID NO: 242. In other aspects ofthis embodiment, a HSV-1 VP 22 translocating sequence comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, or 10 contiguous amino acid deletions relative toSEQ ID NO: 242. In still other aspects of this embodiment, a HSV-1 VP 22translocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10 contiguousamino acid additions relative to SEQ ID NO: 242. In other aspects ofthis embodiment, a HSV-1 VP 22 translocating sequence comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, or 10 contiguous amino acid additions relative toSEQ ID NO: 242.

In another embodiment, a binding domain comprises a translocatingsequence comprises a Penetratin translocating sequence. In anotherembodiment, a binding domain comprises a translocating sequence derivedfrom a Penetratin translocating sequence. In an aspect of thisembodiment, a binding domain comprises a Penetratin translocatingsequence comprises SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO:250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253. In another aspectof this embodiment, a binding domain comprises a Penetratintranslocating sequence derived from SEQ ID NO: 243, SEQ ID NO: 244, SEQID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO:249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252 or SEQ ID NO: 253.

In other aspects of this embodiment, a Penetratin translocating sequencecomprises a polypeptide having, e.g., at least 70% amino acid identitywith SEQ ID NO: 243, at least 75% amino acid identity with SEQ ID NO:243, at least 80% amino acid identity with SEQ ID NO: 243, at least 85%amino acid identity with SEQ ID NO: 243, at least 90% amino acididentity with SEQ ID NO: 243 or at least 95% amino acid identity withSEQ ID NO: 243. In yet other aspects of this embodiment, a Penetratintranslocating sequence comprises a polypeptide having, e.g., at most 70%amino acid identity with SEQ ID NO: 243, at most 75% amino acid identitywith SEQ ID NO: 243, at most 80% amino acid identity with SEQ ID NO:243, at most 85% amino acid identity with SEQ ID NO: 243, at most 90%amino acid identity with SEQ ID NO: 243 or at most 95% amino acididentity with SEQ ID NO: 243.

In other aspects of this embodiment, a Penetratin translocating sequencecomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 243. In other aspects of thisembodiment, a Penetratin translocating sequence comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, or 10 non-contiguous amino acid substitutions relative to SEQ IDNO: 243. In yet other aspects of this embodiment, a Penetratintranslocating sequence comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid deletions relative to SEQ ID NO: 243. In otheraspects of this embodiment, a Penetratin translocating sequencecomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, or 10 non-contiguous amino acid deletionsrelative to SEQ ID NO: 243. In still other aspects of this embodiment, aPenetratin translocating sequence comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine, or 10non-contiguous amino acid additions relative to SEQ ID NO: 243. In otheraspects of this embodiment, a Penetratin translocating sequencecomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, or 10 non-contiguous amino acid additionsrelative to SEQ ID NO: 243.

In other aspects of this embodiment, a Penetratin translocating sequencecomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, or 10 contiguous amino acid substitutionsrelative to SEQ ID NO: 243. In other aspects of this embodiment, aPenetratin translocating sequence comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, or 10contiguous amino acid substitutions relative to SEQ ID NO: 243. In yetother aspects of this embodiment, a Penetratin translocating sequencecomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, or 10 contiguous amino acid deletionsrelative to SEQ ID NO: 243. In other aspects of this embodiment, aPenetratin translocating sequence comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, or 10contiguous amino acid deletions relative to SEQ ID NO: 243. In stillother aspects of this embodiment, a Penetratin translocating sequencecomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, or 10 contiguous amino acid additionsrelative to SEQ ID NO: 243. In other aspects of this embodiment, aPenetratin translocating sequence comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, or 10contiguous amino acid additions relative to SEQ ID NO: 243.

Another example of a binding domain, includes, without limitation, anantibody to a coated pit protein, such as, e.g., a clatherin antibodyand an Adaptor Protein-2 (adaptin) antibody; an antibody to acaveolae-associated protein, such as, e.g., a caveolin-1 antibody and aGPI-linked receptor protein antibody.

One major route of receptor-mediated endocytosis is by means ofendocytotic coated pits. These specialized sites on the plasma membraneof eukaryotic cells are responsible for internalization of many cellsurface receptors, usually after ligand binding. The coated pits servein part to concentrate the receptors for internalization within farfewer endosomes than would be the case if each receptor wereindividually internalized. Coated pits consist largely of two majorproteins: clatherin and the adaptor protein (adaptin) Adaptor Protein-2(AP-2). Assembly of the coated pit is thought to be initiated by thebinding of AP-2 molecules to a receptor or ligand-docking site in themembrane. The receptor:AP-2 complex then recruits the structural proteinclatherin.

Sorting signals present in many membrane proteins, such as cell surfacereceptors, provide for recognition and binding of AP-2 to these proteinsas the initial step of coated pit formation. One common sorting signalis the amino acid sequence YXXΦ (wherein Φ is an amino acid with a bulkyhydrophobic side chain, such as leucine, phenylalanine, methionine andvaline), which provides a signal for rapid internalization. This signalis recognized by and binds to the μ2 subunit of AP-2, and mediates rapidinternalization by mammalian cells. Preferably, the signal is presention such a way as to be displayed on the cytosolic side of the membrane.See e.g., Bonifacino et al., J. Cell Biol. 145: 923 (May 31, 1999).

At least one binding domain of a multivalent Clostridial toxin of thepresent invention may comprise at least one sorting signal for anadaptin, preferably AP-2. Preferably, the binding site comprising thissorting signal is present in addition to another distinct binding domainwhich will directly bind a cell surface receptor.

In certain embodiments, a multivalent Clostridial toxin of the presentinvention comprising two or more pancreatic acinar cell targetingmoieties may be administered to treat pancreatitis; the use of CCK formsand derivatives capable of selective binding to CCK receptors ofpancreatic acinar cells is disclosed in U.S. Pat. No. 6,843,998. In onepreferred embodiment of this aspect of the present invention, two ormore binding moieties of the present invention are independently a CCKor CCK derivative.

In another embodiment of the invention for treatment of, e.g., acute orchronic pancreatitis, at least one binding moiety of the multivalentClostridial toxin is a CCK or CCK derivative, and at least oneadditional binding region comprises a region of a glycosylphosphatylinositol (GPI)-linked membrane protein that directly orindirectly binds caveolin-1 binding. Alternatively, the additionalbinding region may comprise a ligand capable of binding a GPI-linkedmembrane protein, particularly a membrane associated cell surfacereceptor, as the CCK receptor, like the TNF and other known receptors,is known to be concentrated and recruited for internalization bycaveolae.

Caveolae are discrete membrane domains present in many cell types; theyare not thought to be present in neural cells. Caveolae are comprisedlargely of detergent-insoluble lipids and lipid associated proteins,including a major protein, caveolin-1. Caveolae are also known torecruit and concentrate GPI-linked membrane proteins, a family ofmembrane proteins that includes cell surface receptors. Further,caveolae appear to participate in a non clatherin-associated form ofendocytosis, and experiments have been reported in which gene transfervectors have been directed to GPI-linked receptor proteins to facilitategene transfer.

Thus, for example, the urokinase plasminogen activator receptor (uPAR)is a GPI-linked membrane protein that appears to facilitate endocytosisupon binding of its cognate ligand urokinase plasminigen activator(uPA). See e.g., Drapkin et al., J. CLIN. INVEST. 105: 589-596 (Mar. 1,2000), incorporated by reference as part of this specification. uPA is a55 KDa protein secreted by alveolar epithelial cells that cleavesplasminogen to plasmin and is known to degrade the extracellular matrixin alveoli. Targeting such cells, which also display the uPAR, with themultivalent Clostridial toxin of the present invention comprising, forexample, two or more binding domains comprising a uPAR-binding regionderived from, for example, uPA or an anti-uPAR antibody may be oftherapeutic use is the treatment of respiratory and lung diseases suchas e.g., cystic fibrosis, since the ligand-bound receptor is subject toendocytosis in a ligand dependent manner. Thus, for example, a patentmay be administered a mist or dispersion comprising such multivalentClostridial toxin as a delivery mechanism.

In certain embodiments the target cell may comprise a cell comprising amembrane protein that displays an antibody variable region at the cellsurface. In such a case the multivalent Clostridial toxin comprises twoor more binding domains, with at least one binding domain comprising anantigen able to selectively bind the antibody variable region. In apreferred embodiment, the multivalent Clostridial toxin may comprisemore than one binding region comprising an antigen able to selectivelybind to said antibody variable region.

Alternatively, the multivalent Clostridial toxin of the presentinvention (comprising more than one binding domain), may have at leastone binding domain comprising an antigen-binding portion of an antibodyH or L chain, wherein the antigen comprises a cell surface marker for atarget cell. For example, and without limitation, the multivalentClostridial toxin of the present invention may comprise at least onebinding domain comprising an antibody variable region that selectivelybinds to an eosinophil cell surface marker (for example, the cellsurface markers CD 44 and CD 69), thereby aiding in alleviating thesymptoms of allergy. The nomenclature “CD” is derived from the use ofmonoclonal antibodies directed towards a given cell type, and stands for“Cluster of Differentiation”.

In another example, the multivalent Clostridial toxin derivative may beused to inhibit or decrease the rate of infection of T helper cells bythe human immunodeficiency virus (HIV). T helper cells are commonlyidentified by virtue of their display of the cell surface marker CD4.The HIV virus uses the CD4 marker to gain entry into and thereby infectT helper cells, and appears to employ viral fusion proteins sharingremarkable similarity to the SNARE system to invade cells through amembrane fusion mechanism. See e.g., Duman & Forte, AM. J. PHYSIOL. CELLPHYSIOL. 285: C237-C249 (2003). A multivalent Clostridial toxin of thepresent invention comprising at least one, and preferably two or more,anti-CD4 antibody domain or similar CD4 “addressable” binding domainwould permit the neurotoxin protease domain to deny the virus use of theSNARE proteins for entry and/or exit of the cell. Moreover, alterationof the toxin proteolytic domain to selectively recognize and cleave oneor more of the HIV viral fusion proteins, using such techniques asdirected evolution, site directed mutagenesis or other well knownmolecular biology methods, could provide a method of reducing the extentof, or eliminating, HIV infection.

In another aspect of the invention, a multivalent Clostridial toxincomprises, in part, a protease cleavage site. As used herein, the term“protease cleavage site” means a scissile bond together with adjacent ornon-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by a protease under conditions suitablefor protease activity. A protease cleavage site can be an endogenousprotease cleavage site or an exogenous protease cleavage site. Onefunction of the protease cleavage site may be to convert thesingle-chain polypeptide form of a Clostridial toxin into the di-chainform. Another role of a protease cleavage site may be change theconformational structure of a binding domain, thereby facilitating thebinding domain's ability to bind to its cognate receptor, e.g., abinding domain may function only as a dimer, like the binding domainsfrom the TGFβ superfamily; or exposing the amino terminal end of abinding domain, like the binding domains of opiod family members.Likewise, the location and kind of protease cleavage site may becritical because certain targeting domains require a free amino-terminalor carboxyl-terminal amino acid. For example, when a targeting domain isplaced between two other domains, one criterion for selection of aprotease cleavage site could be whether the protease that cleaves itssite leaves a flush cut, exposing the free amino-terminal orcarboxyl-terminal of the altered targeting domain necessary forselective binding of the targeting domain to its receptor. The selectionand placement of a protease cleavage site is well known in the art.

It is envisioned that a multivalent Clostridial toxin may comprise oneprotease cleavage site. It is also envisioned that a multivalentClostridial toxin may comprise a plurality of protease cleavage sites.Thus. in an embodiment, a multivalent Clostridial toxin may comprise oneprotease cleavage site. In another embodiment, a multivalent Clostridialtoxin may comprise a plurality protease cleavage site. In aspects ofthis embodiment, a multivalent Clostridial toxin may comprise, e.g., atleast one protease cleavage site, at least two protease cleavage sites,at least three protease cleavage sites, at least four protease cleavagesites, or at least five protease cleavage sites. In other aspects ofthis embodiment, a multivalent Clostridial toxin may comprise, e.g., atmost one protease cleavage site, at most two protease cleavage sites, atmost three protease cleavage sites, at most four protease cleavagesites, or at most five protease cleavage sites. In still other aspectsof this embodiment, a multivalent Clostridial toxin may comprise, e.g.,one protease cleavage site, two protease cleavage sites, three proteasecleavage sites, four protease cleavage sites, or five protease cleavagesites.

It is envisioned that a multivalent Clostridial toxin can comprise anendogenous protease cleavage. As used herein, the term “endogenousprotease cleavage site” is synonymous with “di-chain loop proteasecleavage site,” “naturally occurring Clostridial toxin di-chain loopprotease cleavage site” or “naturally occurring Clostridial toxinprotease cleavage site” and means a naturally occurring Clostridialtoxin protease cleavage site found within the di-chain loop region of anaturally occurring Clostridial toxin and includes, without limitation,naturally occurring Clostridial toxin di-chain loop protease cleavagesite variants, such as, e.g., Clostridial toxin di-chain loop proteasecleavage site isoforms and Clostridial toxin di-chain loop proteasecleavage site subtypes. Non-limiting examples of an endogenous proteasecleavage site, include, e.g., a BoNT/A di-chain loop protease cleavagesite, a BoNT/B di-chain loop protease cleavage site, a BoNT/C1 di-chainloop protease cleavage site, a BoNT/D di-chain loop protease cleavagesite, a BoNT/E di-chain loop protease cleavage site, a BoNT/F di-chainloop protease cleavage site, a BoNT/G di-chain loop protease cleavagesite and a TeNT di-chain loop protease cleavage site.

As mentioned above, Clostridial toxins are translated as a single-chainpolypeptide of approximately 150 kDa that is subsequently cleaved byproteolytic scission within a disulfide loop by a naturally-occurringprotease. This posttranslational processing yields a di-chain moleculecomprising an approximately 50 kDa light chain (LC) and an approximately100 kDa heavy chain (HC) held together by a single disulphide bond andnoncovalent interactions. While the identity of the protease iscurrently unknown, the di-chain loop protease cleavage site for manyClostridial toxins has been proposed. In BoNTs, cleavage at K448-A449converts the single polypeptide form of BoNT/A into the di-chain form;cleavage at K441-A442 converts the single polypeptide form of BoNT/Binto the di-chain form; cleavage at K449-T450 converts the singlepolypeptide form of BoNT/C1 into the di-chain form; cleavage atR445-D446 converts the single polypeptide form of BoNT/D into thedi-chain form; cleavage at R422-K423 converts the single polypeptideform of BoNT/E into the di-chain form; cleavage at K439-A440 convertsthe single polypeptide form of BoNT/F into the di-chain form; andcleavage at K446-S447 converts the single polypeptide form of BoNT/Ginto the di-chain form. Proteolytic cleavage of the single polypeptideform of TeNT at A457-S458 results in the di-chain form. Such a di-chainloop protease cleavage site is operably-linked in-frame to a multivalentClostridial toxin as a fusion protein.

However, it should also be noted that additional cleavage sites withinthe di-chain loop also appear to be cleaved resulting in the generationof a small peptide fragment being lost. As a non-limiting example,BoNT/A single-chain polypeptide cleave ultimately results in the loss ofa ten amino acid fragment within the di-chain loop. Thus, in BoNTs,cleavage at S441-L442 converts the single polypeptide form of BoNT/Ainto the di-chain form; cleavage at G444-1445 converts the singlepolypeptide form of BoNT/B into the di-chain form; cleavage at S445-L446converts the single polypeptide form of BoNT/C1 into the di-chain form;cleavage at K442-N443 converts the single polypeptide form of BoNT/Dinto the di-chain form; cleavage at K419-G420 converts the singlepolypeptide form of BoNT/E into the di-chain form; cleavage at K423-S424converts the single polypeptide form of BoNT/E into the di-chain form;cleavage at K436-G437 converts the single polypeptide form of BoNT/Finto the di-chain form; cleavage at T444-G445 converts the singlepolypeptide form of BoNT/G into the di-chain form; and cleavage atE448-Q449 converts the single polypeptide form of BoNT/G into thedi-chain form.

As used herein, the term “di-chain loop region” means the amino acidsequence of a Clostridial toxin containing a protease cleavage site usedto convert the single-chain form of a Clostridial toxin into thedi-chain form. Non-limiting examples of a Clostridial toxin di-chainloop region, include, a di-chain loop region of BoNT/A comprising aminoacids 430-454 of SEQ ID NO: 1; a di-chain loop region of BoNT/Bcomprising amino acids 437-446 of SEQ ID NO: 2; a di-chain loop regionof BoNT/C1 comprising amino acids 437-453 of SEQ ID NO: 3; a di-chainloop region of BoNT/D comprising amino acids 437-450 of SEQ ID NO: 4; adi-chain loop region of BoNT/E comprising amino acids 412-426 of SEQ IDNO: 5; a di-chain loop region of BoNT/F comprising amino acids 429-445of SEQ ID NO: 6; a di-chain loop region of BoNT/G comprising amino acids436-450 of SEQ ID NO: 7; and a di-chain loop region of TeNT comprisingamino acids 439-467 of SEQ ID NO: 8 (Table 9).

TABLE 9 Di-chain Loop Region of Clostridial Toxins Di-chain Loop RegionSEQ Containing the Naturally- ID Light Chain occurring Protease HeavyChain Toxin NO: Region Cleavage Site Region BoNT/A 1NMNFTKLKNFTGLFEFYKLL CVRGIITSKTKSLDKGYNK*----ALNDLC IKVNNWDL BoNT/B 2 KQAYEEISKEHLAVYKIQM CKSVK*-------------------APGIC IDVDNEDL BoNT/C1 3  PALRKVNPENMLYLFTKF CHKAIDGRSLYNK*------------TLDC RELLVKNTDL BoNT/D 4  PALQKLSSESVVDLFTKV CLRLTKNSR*---------------DDSTC IKVKNNRL BoNT/E 5   IITPITGRGLVKKIIRF CKNIVSVKGIR*--------------KSIC IEINNGEL BoNT/F 6   IIDSIPDKGLVEKIVKF CKSVIPRKGTK*------------APPRLC IRVNNSEL BoNT/G 7 KEAYEEISLEHLVIYRIAM CKPVMYKNTGK*--------------SEQC IIVNNEDL TeNT 8 TNAFRNVDGSGLVSKLIGL CKKIIPPTNIRENLYNRTA*SLTDLGGELC IKIKNEDL The aminoacid sequence displayed are as follows: BoNT/A, residues 325-462 of SEQID No: 1; BoNT/B, residues 332-454 of SEQ ID No: 2; BoNT/C1, residues334-463 of SEQ ID No: 3; BoNT/D, residues 334-458 of SEQ ID No: 4;BoNT/E, residues 311-434 of SEQ ID No: 5; BoNT/F, residues 328-453 ofSEQ ID No: 6; BoNT/G, residues 331-458 of SEQ ID No: 7; and TeNT,residues 334-474 of SEQ ID No: 8. An asterisks (*) indicates a peptidebond that is cleaved by a Clostridial toxin protease.

Thus, in an embodiment, a multivalent Clostridial toxin comprising aprotease cleavage site comprises an endogenous protease cleavage site.In aspects of this embodiment, an endogenous protease cleavage sitecomprises, e.g., a BoNT/A di-chain loop protease cleavage site, a BoNT/Bdi-chain loop protease cleavage site, a BoNT/C1 di-chain loop proteasecleavage site, a BoNT/D di-chain loop protease cleavage site, a BoNT/Edi-chain loop protease cleavage site, a BoNT/F di-chain loop proteasecleavage site, a BoNT/G di-chain loop protease cleavage site or a TeNTdi-chain loop protease cleavage site.

In other aspects of this embodiment, an endogenous protease cleavagesite comprises, e.g., a di-chain loop region of BoNT/A comprising aminoacids 430-454 of SEQ ID NO: 1; a di-chain loop region of BoNT/Bcomprising amino acids 437-446 of SEQ ID NO: 2; a di-chain loop regionof BoNT/C1 comprising amino acids 437-453 of SEQ ID NO: 3; a di-chainloop region of BoNT/D comprising amino acids 437-450 of SEQ ID NO: 4; adi-chain loop region of BoNT/E comprising amino acids 412-426 of SEQ IDNO: 5; a di-chain loop region of BoNT/F comprising amino acids 429-445of SEQ ID NO: 6; a di-chain loop region of BoNT/G comprising amino acids436-450 of SEQ ID NO: 7; or a di-chain loop region of TeNT comprisingamino acids 439-467 of SEQ ID NO: 8.

It is also envisioned that a multivalent Clostridial toxin can comprisean exogenous protease cleavage. As used herein, the term “exogenousprotease cleavage site” is synonymous with a “non-naturally occurringClostridial toxin protease cleavage site” and means a protease cleavagesite that is not normally present in a di-chain loop region from anaturally occurring Clostridial toxin. Non-limiting examples ofexogenous protease cleavage sites include, e.g., an enterokinasecleavage site (Table 10); a Thrombin cleavage site (Table 10); a FactorXa cleavage site (Table 10); a human rhinovirus 3C protease cleavagesite (Table 10); a tobacco etch virus (TEV) protease cleavage site(Table 10); a dipeptidyl aminopeptidase cleavage site; a smallubiquitin-like modifier (SUMO)/ubiquitin-like protein-1(ULP-1) proteasecleavage site, such as, e.g., MADSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (SEQ ID. NO:271); and a Clostridial toxin substrate cleavage site.

As mentioned above, a Clostridial toxin is converted from a singlepolypeptide form into a di-chain molecule by proteolytic cleavage. Whilethe naturally-occurring protease is currently not known, cleavage occurswithin the di-chain loop region between the two cysteine residues thatform the disulfide bridge (see Table 9). Replacement of an endogenousprotease cleavage site with an exogenous protease cleavage site willenable cleavage of a multivalent Clostridial toxin disclosed in thepresent specification when expressed in an organism that does notproduce the naturally-occurring protease used to cleave the di-chainloop region of a toxin. Similarly, an addition of an exogenous proteasecleavage site in the di-chain loop region will also enable cleavage of amultivalent Clostridial toxin disclosed in the present specificationwhen expressed in an organism that does not produce thenaturally-occurring protease used to cleave the di-chain loop region ofa toxin. Furthermore, cleavage at an exogenous protease cleavage sitecan facilitate a change in the conformational structure of a bindingdomain, thereby facilitating the binding domain's ability to bind to itscognate receptor, e.g., a binding domain may function only as a dimer,like the binding domains from the TGFβ superfamily; or exposing theamino terminal end of a binding domain, like the binding domains ofopiod family members.

TABLE 10 Exogenous Protease Cleavage Sites Protease CleavageNon-limiting SEQ ID Site Consensus Sequence Examples NO: Bovineenterokinase DDDDK* DDDDK* 254 Tobacco Etch Virus E P⁵ P⁴YP²Q*(G/S),ENLYFQ*G 255 (TEV) where P², P⁴ and P⁵ can be ENLYFQ*S 256 any aminoacid ENIYTQ*G 257 ENIYTQ*S 258 ENIYLQ*G 259 ENIYLQ*S 260 ENVYFQ*G 261ENVYSQ*S 262 ENVYSQ*G 263 ENVYSQ*S 264 Human Rhinovirus 3C P⁵P⁴LFQ*GPEALFQ*GP 265 where P⁴ is G, A, V, L, I, EVLFQ*GP 266 M, S or T and P⁵can any ELLFQ*GP 267 amino acid, with D or E DALFQ*GP 268 preferred.DVLFQ*GP 269 DLLFQ*GP 270 SUMO/ULP-1 Tertiary structure polypeptide-G*271 Thrombin P³P²(R/K)*P^(1′), GVR*G 272 where P³ is any amino acidSAR*G 273 and P² or P^(1′ )is G with the SLR*G 274 other position beingany DGR*I 275 amino acid QGK*I 276 Thrombin P⁴P³(R/K)*P^(1′)P^(2′)LVPR*GS 277 where P^(1′ )and P^(2′ )can be any LVPK*GS 278 amino acidexcept for acidic FIPR*TF 279 amino acids like D or E; and VLPR*SF 280P³ and P⁴ are hydrophobic IVPR*SF 281 amino acids like F, L, I, Y,IVPR*GY 282 W, V, M, P, C or A VVPR*GV 283 VLPR*LI 284 VMPR*SL 285MFPR*SL 286 Coagulation Factor I(E/D)GR* IDGR* 287 Xa IEGR* 288 Anasterisks (*) indicates the peptide bond that is cleaved by theindicated protease.

It is envisioned that an exogenous protease cleavage site of any and alllengths can be useful in aspects of the present invention with theproviso that the exogenous protease cleavage site is capable of beingcleaved by its respective protease. Thus, in aspects of this embodiment,an exogenous protease cleavage site can be, e.g., at least 6 amino acidsin length, at least 7 amino acids in length, at least 8 amino acids inlength, at least 9 amino acids in length, at least 10 amino acids inlength, at least 15 amino acids in length, at least 20 amino acids inlength, at least 25 amino acids in length, at least 30 amino acids inlength, at least 40 amino acids in length, at least 50 amino acids inlength or at least 60 amino acids in length. In other aspects of thisembodiment, an exogenous protease cleavage site can be, e.g., at most 6amino acids in length, at most 7 amino acids in length, at most 8 aminoacids in length, at most 9 amino acids in length, at most 10 amino acidsin length, at most 15 amino acids in length, at most 20 amino acids inlength, at most 25 amino acids in length, at most 30 amino acids inlength, at most 40 amino acids in length, at most 50 amino acids inlength or at most 60 amino acids in length.

In aspects of this embodiment, a di-chain loop region can be modified tosubstitute a naturally-occurring protease cleavage site for an exogenousprotease cleavage site. In this type of modification, thenaturally-occurring protease cleavage site is made inoperable and thuscan not be cleaved by its protease. Only the exogenous protease cleavagesite can be cleaved by its corresponding exogenous protease. In thistype of modification, the exogenous protease site is operably-linkedin-frame to a multivalent Clostridial toxin as a fusion protein and thesite can be cleaved by its respective exogenous protease. As anon-limiting example, a single-chain modified BoNT/A comprising anexogenous protease cleavage site in the di-chain loop region can becleaved by its respective exogenous protease to produce the di-chainform of the toxin.

In other aspects of this embodiment, a di-chain loop region can bemodified to include an exogenous protease cleavage site in addition tothe naturally-occurring protease cleavage site. In this type ofmodification, both cleavage sites are operably-linked in-frame to amultivalent Clostridial toxin as a fusion protein and both sites can becleaved by their respective proteases. As a non-limiting example, asingle-chain modified BoNT/A that comprises a di-chain loop containingboth the naturally-occurring BoNT/A di-chain loop protease cleavage siteand an exogenous protease cleavage site can be cleaved by either thenaturally occurring di-chain loop protease or by the appropriateexogenous protease to produce the di-chain form of the toxin.

A naturally-occurring protease cleavage site can be made inoperable byaltering at least the two amino acids flanking the peptide bond cleavedby the naturally-occurring di-chain loop protease. More extensivealterations can be made, with the proviso that the two cysteine residuesof the di-chain loop region remain intact and can still form thedisulfide bridge. Non-limiting examples of an amino acid alterationinclude deletion of an amino acid or replacement of the original aminoacid with a different amino acid. Thus, in one embodiment, anaturally-occurring protease cleavage site is made inoperable byaltering the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease. In other aspects of this embodiment, anaturally-occurring protease cleavage site is made inoperable byaltering, e.g., at least three amino acids including the two amino acidsflanking the peptide bond cleaved by a naturally-occurring protease; atleast four amino acids including the two amino acids flanking thepeptide bond cleaved by a naturally-occurring protease; at least fiveamino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease; at least six amino acidsincluding the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least seven amino acids including thetwo amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least eight amino acids including thetwo amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least nine amino acids including thetwo amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least ten amino acids including the twoamino acids flanking the peptide bond cleaved by a naturally-occurringprotease; at least 15 amino acids including the two amino acids flankingthe peptide bond cleaved by a naturally-occurring protease; or at least20 amino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease.

In still other aspects of this embodiment, a naturally-occurringdi-chain protease cleavage site is made inoperable by altering, e.g., atmost three amino acids including the two amino acids flanking thepeptide bond cleaved by a naturally-occurring protease; at most fouramino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease; at most five amino acidsincluding the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at most six amino acids including the twoamino acids flanking the peptide bond cleaved by a naturally-occurringprotease; at most seven amino acids including the two amino acidsflanking the peptide bond cleaved by a naturally-occurring protease; atmost eight amino acids including the two amino acids flanking thepeptide bond cleaved by a naturally-occurring protease; at most nineamino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease; at most ten amino acidsincluding the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at most 15 amino acids including the twoamino acids flanking the peptide bond cleaved by a naturally-occurringprotease; or at most 20 amino acids including the two amino acidsflanking the peptide bond cleaved by a naturally-occurring protease.

In an embodiment, an exogenous protease cleavage site is located withinthe di-chain loop of a multivalent Clostridial toxin. In aspects of thisembodiment, a multivalent Clostridial toxin comprises an exogenousprotease cleavage site comprises, e.g., a bovine enterokinase proteasecleavage site, a Tobacco Etch Virus protease cleavage site, a HumanRhinovirus 3C protease cleavage site, a SUMO/ULP-1 protease cleavagesite, a Thrombin protease cleavage site or a Factor Xa protease cleavagesite. In other aspects of this embodiment, an exogenous proteasecleavage site is located within the di-chain loop of, e.g., a modifiedBoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, amodified BoNT/E, a modified BoNT/F, a modified BoNT/G or a modifiedTeNT.

In an aspect of this embodiment, an exogenous protease cleavage site canbe, e.g., a bovine enterokinase cleavage site is located within thedi-chain loop of a multivalent Clostridial toxin. In other aspects ofthe embodiment, an exogenous protease cleavage site can be, e.g., abovine enterokinase protease cleavage site located within the di-chainloop of a multivalent Clostridial toxin comprises SEQ ID NO: 254. Isstill other aspects of this embodiment, a bovine enterokinase proteasecleavage site is located within the di-chain loop of, e.g., a modifiedBoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, amodified BoNT/E, a modified BoNT/F, a modified BoNT/G or a modifiedTeNT.

In another aspect of this embodiment, an exogenous protease cleavagesite can be, e.g., a Tobacco Etch Virus protease cleavage site islocated within the di-chain loop of a multivalent Clostridial toxin. Inother aspects of the embodiment, an exogenous protease cleavage site canbe, e.g., a Tobacco Etch Virus protease cleavage site located within thedi-chain loop of a multivalent Clostridial toxin comprises SEQ ID NO:255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263 or SEQ ID NO:264. Is still other aspects of this embodiment, a Tobacco Etch Virusprotease cleavage site is located within the di-chain loop of, e.g., amodified BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modifiedBoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G or amodified TeNT.

In still another aspect of this embodiment, an exogenous proteasecleavage site can be, e.g., a Human Rhinovirus 3C protease cleavage siteis located within the di-chain loop of a multivalent Clostridial toxin.In other aspects of the embodiment, an exogenous protease cleavage sitecan be, e.g., a Human Rhinovirus 3C protease cleavage site locatedwithin the di-chain loop of a multivalent Clostridial toxin comprisesSEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ IDNO: 269 or SEQ ID NO: 270. Is still other aspects of this embodiment, aHuman Rhinovirus 3C protease cleavage site is located within thedi-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B, a modifiedBoNT/C1, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, amodified BoNT/G or a modified TeNT.

In yet another aspect of this embodiment, an exogenous protease cleavagesite can be, e.g., a SUMO/ULP-1 protease cleavage site is located withinthe di-chain loop of a multivalent Clostridial toxin. In other aspectsof the embodiment, an exogenous protease cleavage site can be, e.g., aSUMO/ULP-1 protease cleavage site located within the di-chain loop of amultivalent Clostridial toxin comprises SEQ ID NO: 271. Is still otheraspects of this embodiment, a SUMO/ULP-1 protease cleavage site islocated within the di-chain loop of, e.g., a modified BoNT/A, a modifiedBoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, amodified BoNT/F, a modified BoNT/G or a modified TeNT.

In a further aspect of this embodiment, an exogenous protease cleavagesite can be, e.g., a Thrombin protease cleavage site is located withinthe di-chain loop of a multivalent Clostridial toxin. In other aspectsof the embodiment, an exogenous protease cleavage site can be, e.g., aThrombin protease cleavage site located within the di-chain loop of amultivalent Clostridial toxin comprises SEQ ID NO: 272, SEQ ID NO: 273,SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ IDNO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282,SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285 or SEQ ID NO: 286. Isstill other aspects of this embodiment, a Thrombin protease cleavagesite is located within the di-chain loop of, e.g., a modified BoNT/A, amodified BoNT/B, a modified BoNT/C1, a modified BoNT/D, a modifiedBoNT/E, a modified BoNT/F, a modified BoNT/G or a modified TeNT.

In another aspect of this embodiment, an exogenous protease cleavagesite can be, e.g., a Coagulation Factor Xa protease cleavage site islocated within the di-chain loop of a multivalent Clostridial toxin. Inother aspects of the embodiment, an exogenous protease cleavage site canbe, e.g., a Coagulation Factor Xa protease cleavage site located withinthe di-chain loop of a multivalent Clostridial toxin comprises SEQ IDNO: 287 or SEQ ID NO: 288. Is still other aspects of this embodiment, aCoagulation Factor Xa protease cleavage site is located within thedi-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B, a modifiedBoNT/C1, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, amodified BoNT/G or a modified TeNT.

In another embodiment, an exogenous protease site comprises aClostridial toxin substrate cleavage site. As used herein, the term“Clostridial toxin substrate cleavage site” means a scissile bondtogether with adjacent or non-adjacent recognition elements, or both,sufficient for detectable proteolysis at the scissile bond by aClostridial toxin under conditions suitable for Clostridial toxinprotease activity. By definition, a Clostridial toxin substrate cleavagesite is susceptible to cleavage by at least one Clostridial toxin underconditions suitable for Clostridial toxin protease activity.Non-limiting examples of Clostridial toxin substrate cleavage site aredisclosed in, e.g., Steward, L. E. et al., Self-Activating ClostridialToxins, U.S. Patent Application 60/718,616 (Sep. 19, 2005).

It is understood that a multivalent Clostridial toxin disclosed in thepresent specification can optionally include one or more additionalcomponents. As a non-limiting example of an optional component, amultivalent Clostridial toxin can further comprise a flexible regioncomprising a flexible spacer. Non-limiting examples of a flexible spacerinclude, e.g., a G-spacer GGGGS (SEQ ID NO: 289) or an A-spacer EAAAK(SEQ ID NO: 290). A flexible region comprising flexible spacers can beused to adjust the length of a polypeptide region in order to optimize acharacteristic, attribute or property of a polypeptide. Such a flexibleregion is operably-linked in-frame to the multivalent Clostridial toxinas a fusion protein. As a non-limiting example, a polypeptide regioncomprising one or more flexible spacers in tandem can be use to betterexpose a protease cleavage site thereby facilitating cleavage of thatsite by a protease. As another non-limiting example, a polypeptideregion comprising one or more flexible spacers in tandem can be use tobetter present an enhanced targeting domain, thereby facilitating thebinding of that enhanced targeting domain to its receptor.

Thus, in an embodiment, a multivalent Clostridial toxin disclosed in thepresent specification can further comprise a flexible region comprisinga flexible spacer. In another embodiment, a multivalent Clostridialtoxin disclosed in the present specification can further compriseflexible region comprising a plurality of flexible spacers in tandem. Inaspects of this embodiment, a flexible region can comprise in tandem,e.g., at least 1 G-spacer, at least 2 G-spacers, at least 3 G-spacers,at least 4 G-spacers or at least 5 G-spacers. In other aspects of thisembodiment, a flexible region can comprise in tandem, e.g., at most 1G-spacer, at most 2 G-spacers, at most 3 G-spacers, at most 4 G-spacersor at most 5 G-spacers. In still other aspects of this embodiment, aflexible region can comprise in tandem, e.g., at least 1 A-spacer, atleast 2 A-spacers, at least 3 A-spacers, at least 4 A-spacers or atleast 5 A-spacers. In still other aspects of this embodiment, a flexibleregion can comprise in tandem, e.g., at most 1 A-spacer, at most 2A-spacers, at most 3 A-spacers, at most 4 A-spacers or at most 5A-spacers. In another aspect of this embodiment, a multivalentClostridial toxin can comprise a flexible region comprising one or morecopies of the same flexible spacers, one or more copies of differentflexible-spacer regions, or any combination thereof.

In aspects of this embodiment, a multivalent Clostridial toxincomprising a flexible spacer can be, e.g., a modified BoNT/A, a modifiedBoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, amodified BoNT/F, a modified BoNT/G or a modified TeNT.

It is envisioned that a multivalent Clostridial toxin disclosed in thepresent specification can comprise a flexible spacer in any and alllocations with the proviso that multivalent Clostridial toxin is capableof performing the intoxication process. In aspects of this embodiment, aflexible spacer is positioned between, e.g., an enzymatic domain and atranslocation domain, an enzymatic domain and a binding domain, anenzymatic domain and a protease cleavage site. In other aspects of thisembodiment, a G-spacer is positioned between, e.g., an enzymatic domainand a translocation domain, an enzymatic domain and a binding domain, anenzymatic domain and a protease cleavage site. In other aspects of thisembodiment, a A-spacer is positioned between, e.g., an enzymatic domainand a translocation domain, an enzymatic domain and a binding domain, anenzymatic domain and a protease cleavage site.

In other aspects of this embodiment, a flexible spacer is positionedbetween, e.g., a binding domain and a translocation domain, a bindingdomain and an enzymatic domain, a binding domain and a protease cleavagesite. In other aspects of this embodiment, a G-spacer is positionedbetween, e.g., a binding domain and a translocation domain, a bindingdomain and an enzymatic domain, a binding domain and a protease cleavagesite. In other aspects of this embodiment, a A-spacer is positionedbetween, e.g., a binding domain and a translocation domain, a bindingdomain and an enzymatic domain, a binding domain and a protease cleavagesite.

In yet other aspects of this embodiment, a flexible spacer is positionedbetween, e.g., a translocation domain and an enzymatic domain, antranslocation domain and a binding domain, an translocation domain and aprotease cleavage site. In other aspects of this embodiment, a G-spaceris positioned between, e.g., a translocation domain and an enzymaticdomain, an translocation domain and a binding domain, an translocationdomain and a protease cleavage site. In other aspects of thisembodiment, a A-spacer is positioned between, e.g., a translocationdomain and an enzymatic domain, an translocation domain and a bindingdomain, a translocation domain and a protease cleavage site.

It is envisioned that a multivalent Clostridial toxin disclosed in thepresent specification can comprise a binding domain 1 and a bindingdomain 2 in any and all locations with the proviso that multivalentClostridial toxin is capable of performing the intoxication process.Non-limiting examples are depicted in FIG. 4), FIG. 5, and FIG. 6. Theenzymatic domain of naturally-occurring Clostridial toxins contains thenative start methionine. Thus, in domain organizations where theenzymatic domain is not in the amino-terminal location an amino acidsequence comprising the start methionine should be placed in front ofthe amino-terminal domain. Likewise, where a binding domain is in theamino-terminal position, an amino acid sequence comprising a startmethionine and a protease cleavage site may be operably-linked insituations in which the enhanced targeting domain requires a free aminoterminus, see, e.g., Shengwen Li et al., Degradable Clostridial Toxins,International Patent Application Publication WO 2006/026780 (Mar. 9,2006). In addition, it is known in the art that when adding apolypeptide that is operably-linked to the amino terminus of anotherpolypeptide comprising the start methionine that the original methionineresidue can be deleted.

Thus, in an embodiment, a multivalent Clostridial toxin can comprise anamino to carboxyl linear organization comprising a binding domain 1, atranslocation domain, a binding domain 2 and an enzymatic domain. In anaspect of this embodiment, a multivalent Clostridial toxin can comprisean amino to carboxyl linear organization comprising a binding domain 1,a translocation domain, a binding domain 2, a protease cleavage site andan enzymatic domain. In another aspect of this embodiment, a multivalentClostridial toxin can comprise an amino to carboxyl linear organizationcomprising a binding domain 1, a translocation domain, a binding domain2, an endogenous protease cleavage site and an enzymatic domain. Inanother aspect of this embodiment, a multivalent Clostridial toxin cancomprise an amino to carboxyl linear organization comprising a bindingdomain 1, a translocation domain, a binding domain 2, an exogenousprotease cleavage site and an enzymatic domain.

In another embodiment, a multivalent Clostridial toxin can comprise anamino to carboxyl linear organization comprising an enzymatic domain, abinding domain 1, a translocation domain and a binding domain 2. In anaspect of this embodiment, a multivalent Clostridial toxin can comprisean amino to carboxyl linear organization comprising an enzymatic domain,a protease cleavage site, a binding domain 1, a translocation domain anda binding domain 2. In another aspect of this embodiment, a multivalentClostridial toxin can comprise an amino to carboxyl linear organizationcomprising an enzymatic domain, an endogenous protease cleavage site, abinding domain 1, a translocation domain and a binding domain 2. Inanother aspect of this embodiment, a multivalent Clostridial toxin cancomprise an amino to carboxyl linear organization comprising anenzymatic domain, an exogenous protease cleavage site, a binding domain1, a translocation domain and a binding domain 2.

In another embodiment, a multivalent Clostridial toxin can comprise anamino to carboxyl linear organization comprising an enzymatic domain, atranslocation domain, a binding domain 1 and a binding domain 2. In anaspect of this embodiment, a multivalent Clostridial toxin can comprisean amino to carboxyl linear organization comprising an enzymatic domain,a protease cleavage site, a translocation domain, a binding domain 1 anda binding domain 2. In another aspect of this embodiment, a multivalentClostridial toxin can comprise an amino to carboxyl linear organizationcomprising an enzymatic domain, an endogenous protease cleavage site, atranslocation domain, a binding domain 1 and a binding domain 2. Inanother aspect of this embodiment, a multivalent Clostridial toxin cancomprise an amino to carboxyl linear organization comprising anenzymatic domain, an exogenous protease cleavage site, a translocationdomain, a binding domain 1 and a binding domain 2.

In another embodiment, a multivalent Clostridial toxin can comprise anamino to carboxyl linear organization comprising an enzymatic domain, atranslocation domain, a binding domain 2 and a binding domain 1. In anaspect of this embodiment, a multivalent Clostridial toxin can comprisean amino to carboxyl linear organization comprising an enzymatic domain,a protease cleavage site, a translocation domain, a binding domain 2 anda binding domain 1. In another aspect of this embodiment, a multivalentClostridial toxin can comprise an amino to carboxyl linear organizationcomprising an enzymatic domain, an endogenous protease cleavage site, atranslocation domain, a binding domain 2 and a binding domain 1. Inanother aspect of this embodiment, a multivalent Clostridial toxin cancomprise an amino to carboxyl linear organization comprising anenzymatic domain, an exogenous protease cleavage site, a translocationdomain, a binding domain 2 and a binding domain 1.

In another embodiment, a multivalent Clostridial toxin can comprise anamino to carboxyl linear organization comprising a binding domain 1, anenzymatic domain, a translocation domain and a binding domain 2. In anaspect of this embodiment, a multivalent Clostridial toxin can comprisean amino to carboxyl linear organization comprising a binding domain 1,an enzymatic domain, a protease cleavage site, a translocation domainand a binding domain 2. In an aspect of this embodiment, a multivalentClostridial toxin can comprise an amino to carboxyl linear organizationcomprising a binding domain 1, an enzymatic domain, an endogenousprotease cleavage site, a translocation domain and a binding domain 2.In an aspect of this embodiment, a multivalent Clostridial toxin cancomprise an amino to carboxyl linear organization comprising a bindingdomain 1, an enzymatic domain, an exogenous protease cleavage site, atranslocation domain and a binding domain 2.

In another embodiment, a multivalent Clostridial toxin can comprise anamino to carboxyl linear organization comprising a translocation domain,a binding domain 2, a binding domain 1 and an enzymatic domain. In anaspect of this embodiment, a multivalent Clostridial toxin can comprisean amino to carboxyl linear organization comprising a translocationdomain, a binding domain 2, a protease cleavage site, a binding domain 1and an enzymatic domain. In another aspect of this embodiment, amultivalent Clostridial toxin can comprise an amino to carboxyl linearorganization comprising a translocation domain, a binding domain 2, anendogenous protease cleavage site, a binding domain 1 and an enzymaticdomain. In another aspect of this embodiment, a multivalent Clostridialtoxin can comprise an amino to carboxyl linear organization comprising atranslocation domain, a binding domain 2, an exogenous protease cleavagesite, a binding domain 1 and an enzymatic domain.

In another embodiment, a multivalent Clostridial toxin can comprise anamino to carboxyl linear organization comprising a translocation domain,a binding domain 2, an enzymatic domain and a binding domain 1. In anaspect of this embodiment, a multivalent Clostridial toxin can comprisean amino to carboxyl linear organization comprising a translocationdomain, a binding domain 2, a protease cleavage site, an enzymaticdomain and a binding domain 1. In another aspect of this embodiment, amultivalent Clostridial toxin can comprise an amino to carboxyl linearorganization comprising a translocation domain, a binding domain 2, anendogenous protease cleavage site, an enzymatic domain and a bindingdomain 1. In another aspect of this embodiment, a multivalentClostridial toxin can comprise an amino to carboxyl linear organizationcomprising a translocation domain, a binding domain 2, an exogenousprotease cleavage site, an enzymatic domain and a binding domain 1.

In another embodiment, a multivalent Clostridial toxin can comprise anamino to carboxyl linear organization comprising a binding domain 2, atranslocation domain, a binding domain 1 and an enzymatic domain. In anaspect of this embodiment, a multivalent Clostridial toxin can comprisean amino to carboxyl linear organization comprising a binding domain 2,a translocation domain, a protease cleavage site, a binding domain 1 andan enzymatic domain. In another aspect of this embodiment, a multivalentClostridial toxin can comprise an amino to carboxyl linear organizationcomprising a binding domain 2, a translocation domain, an endogenouscleavage protease site, a binding domain 1 and an enzymatic domain. Inanother aspect of this embodiment, a multivalent Clostridial toxin cancomprise an amino to carboxyl linear organization comprising a bindingdomain 2, a translocation domain, an exogenous protease cleavage site, abinding domain 1 and an enzymatic domain.

In another embodiment, a multivalent Clostridial toxin can comprise anamino to carboxyl linear organization comprising an enzymatic domain, atranslocation domain, a binding domain 1 and a binding domain 2. In anaspect of this embodiment, a multivalent Clostridial toxin can comprisean amino to carboxyl linear organization comprising an enzymatic domain,a first protease cleavage site, a translocation domain, a binding domain1, a second protease cleavage site, and a binding domain 2. In anotheraspect of this embodiment, a multivalent Clostridial toxin can comprisean amino to carboxyl linear organization comprising an enzymatic domain,a first endogenous protease cleavage site, a translocation domain, abinding domain 1, a second endogenous protease cleavage site, and abinding domain 2. In another aspect of this embodiment, a multivalentClostridial toxin can comprise an amino to carboxyl linear organizationcomprising an enzymatic domain, a first exogenous protease cleavagesite, a translocation domain, a binding domain 1, a second exogenousprotease cleavage site, and a binding domain 2.

In another embodiment, a multivalent Clostridial toxin can comprise anamino to carboxyl linear organization comprising an enzymatic domain, atranslocation domain, a binding domain 2 and a binding domain 1. In anaspect of this embodiment, a multivalent Clostridial toxin can comprisean amino to carboxyl linear organization comprising an enzymatic domain,a first protease cleavage site, a translocation domain, a binding domain2, a second protease cleavage site, and a binding domain 1. In anotheraspect of this embodiment, a multivalent Clostridial toxin can comprisean amino to carboxyl linear organization comprising an enzymatic domain,a first endogenous protease cleavage site, a translocation domain, abinding domain 2, a second endogenous protease cleavage site, and abinding domain 1. In another aspect of this embodiment, a multivalentClostridial toxin can comprise an amino to carboxyl linear organizationcomprising an enzymatic domain, a first exogenous protease cleavagesite, a translocation domain, a binding domain 2, a second exogenousprotease cleavage site, and a binding domain 1.

Aspects of the present invention provide, in part multivalentClostridial toxins. Non-limiting examples of Clostridial toxinmodifications disclosed in the present specification include, e.g.,addition of a binding domain 1, addition of a binding domain 2, additionof a protease cleavage site, rearrangement of the enzymatic,translocation and binding domains and addition of a spacer region. It isunderstood that all such modifications do not substantially affect theability of a multivalent Clostridial toxin to intoxicate a cell. As usedherein, the term “do not substantially affect” means a multivalentClostridial toxin can still execute the overall cellular mechanismwhereby a Clostridial toxin enters a neuron and inhibitsneurotransmitter release and encompasses the binding of a Clostridialtoxin to a low or high affinity receptor complex, the internalization ofthe toxin/receptor complex, the translocation of the Clostridial toxinlight chain into the cytoplasm and the enzymatic modification of aClostridial toxin substrate. In aspects of this embodiment, themultivalent Clostridial toxin is, e.g., at least 10% as toxic as anaturally-occurring Clostridial toxin, at least 20% as toxic as anaturally-occurring Clostridial toxin, at least 30% as toxic as anaturally-occurring Clostridial toxin, at least 40% as toxic as anaturally-occurring Clostridial toxin, at least 50% as toxic as anaturally-occurring Clostridial toxin, at least 60% as toxic as anaturally-occurring Clostridial toxin, at least 70% as toxic as anaturally-occurring Clostridial toxin, at least 80% as toxic as anaturally-occurring Clostridial toxin, at least 90% as toxic as anaturally-occurring Clostridial toxin or at least 95% as toxic as anaturally-occurring Clostridial toxin. In aspects of this embodiment,the multivalent Clostridial toxin is, e.g., at most 10% as toxic as anaturally-occurring Clostridial toxin, at most 20% as toxic as anaturally-occurring Clostridial toxin, at most 30% as toxic as anaturally-occurring Clostridial toxin, at most 40% as toxic as anaturally-occurring Clostridial toxin, at most 50% as toxic as anaturally-occurring Clostridial toxin, at most 60% as toxic as anaturally-occurring Clostridial toxin, at most 70% as toxic as anaturally-occurring Clostridial toxin, at most 80% as toxic as anaturally-occurring Clostridial toxin, at most 90% as toxic as anaturally-occurring Clostridial toxin or at most 95% as toxic as anaturally-occurring Clostridial toxin.

Another aspect of the present invention provides polynucleotidemolecules encoding multivalent Clostridial toxins disclosed in thepresent specification. It is envisioned that any and all multivalentClostridial toxin disclosed in the present specification can be encodedby a polynucleotide molecule.

Aspects of the present invention provide, in part polynucleotidemolecules. As used herein, the term “polynucleotide molecule” issynonymous with “nucleic acid molecule” and means a polymeric form ofnucleotides, such as, e.g., ribonucleotides and deoxyribonucleotides, ofany length. It is envisioned that any and all polynucleotide moleculesthat can encode a multivalent Clostridial toxin disclosed in the presentspecification can be useful, including, without limitationnaturally-occurring and non-naturally-occurring DNA molecules andnaturally-occurring and non-naturally-occurring RNA molecules.Non-limiting examples of naturally-occurring and non-naturally-occurringDNA molecules include single-stranded DNA molecules, double-stranded DNAmolecules, genomic DNA molecules, cDNA molecules, vector constructs,such as, e.g., plasmid constructs, phagmid constructs, bacteriophageconstructs, retroviral constructs and artificial chromosome constructs.Non-limiting examples of naturally-occurring and non-naturally-occurringRNA molecules include single-stranded RNA, double stranded RNA and mRNA.

Well-established molecular biology techniques that may be necessary tomake a polynucleotide molecule encoding a multivalent Clostridial toxindisclosed in the present specification including, but not limited to,procedures involving polymerase chain reaction (PCR) amplification,restriction enzyme reactions, agarose gel electrophoresis, nucleic acidligation, bacterial transformation, nucleic acid purification, nucleicacid sequencing and recombination-based techniques are routineprocedures well within the scope of one skilled in the art and from theteaching herein. Non-limiting examples of specific protocols necessaryto make a polynucleotide molecule encoding a multivalent Clostridialtoxin are described in e.g., MOLECULAR CLONING A LABORATORY MANUAL,supra, (2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Frederick M.Ausubel et al., eds. John Wiley & Sons, 2004). Additionally, a varietyof commercially available products useful for making a polynucleotidemolecule encoding a multivalent Clostridial toxin are widely available.These protocols are routine procedures well within the scope of oneskilled in the art and from the teaching herein.

Another aspect of the present invention provides a method of producing amultivalent Clostridial toxin disclosed in the present specification,such method comprising the step of expressing a polynucleotide moleculeencoding a multivalent Clostridial toxin in a cell. Another aspect ofthe present invention provides a method of producing a multivalentClostridial toxin disclosed in the present specification, such methodcomprising the steps of introducing an expression construct comprising apolynucleotide molecule encoding a multivalent Clostridial toxin into acell and expressing the expression construct in the cell.

The methods disclosed in the present specification include, in part, amultivalent Clostridial toxin. It is envisioned that any and allmultivalent Clostridial toxins disclosed in the present specificationcan be produced using the methods disclosed in the presentspecification. It is also envisioned that any and all polynucleotidemolecules encoding a multivalent Clostridial toxins disclosed in thepresent specification can be useful in producing a multivalentClostridial toxins disclosed in the present specification using themethods disclosed in the present specification.

The methods disclosed in the present specification include, in part, anexpression construct. An expression construct comprises a polynucleotidemolecule disclosed in the present specification operably-linked to anexpression vector useful for expressing the polynucleotide molecule in acell or cell-free extract. A wide variety of expression vectors can beemployed for expressing a polynucleotide molecule encoding a multivalentClostridial toxin, including, without limitation, a viral expressionvector; a prokaryotic expression vector; eukaryotic expression vectors,such as, e.g., a yeast expression vector, an insect expression vectorand a mammalian expression vector; and a cell-free extract expressionvector. It is further understood that expression vectors useful topractice aspects of these methods may include those which express amultivalent Clostridial toxin under control of a constitutive,tissue-specific, cell-specific or inducible promoter element, enhancerelement or both. Non-limiting examples of expression vectors, along withwell-established reagents and conditions for making and using anexpression construct from such expression vectors are readily availablefrom commercial vendors that include, without limitation, BDBiosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, SanDiego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; EMDBiosciences-Novagen, Madison, Wis.; QIAGEN, Inc., Valencia, Calif.; andStratagene, La Jolla, Calif. The selection, making and use of anappropriate expression vector are routine procedures well within thescope of one skilled in the art and from the teachings herein.

Thus, aspects of this embodiment include, without limitation, a viralexpression vector operably-linked to a polynucleotide molecule encodinga multivalent Clostridial toxin; a prokaryotic expression vectoroperably-linked to a polynucleotide molecule encoding a multivalentClostridial toxin; a yeast expression vector operably-linked to apolynucleotide molecule encoding a multivalent Clostridial toxin; aninsect expression vector operably-linked to a polynucleotide moleculeencoding a multivalent Clostridial toxin; and a mammalian expressionvector operably-linked to a polynucleotide molecule encoding amultivalent Clostridial toxin. Other aspects of this embodiment include,without limitation, expression constructs suitable for expressing amultivalent Clostridial toxin disclosed in the present specificationusing a cell-free extract comprising a cell-free extract expressionvector operably linked to a polynucleotide molecule encoding amultivalent Clostridial toxin.

The methods disclosed in the present specification include, in part, acell. It is envisioned that any and all cells can be used. Thus, aspectsof this embodiment include, without limitation, prokaryotic cellsincluding, without limitation, strains of aerobic, microaerophilic,capnophilic, facultative, anaerobic, gram-negative and gram-positivebacterial cells such as those derived from, e.g., Escherichia coli,Bacillus subtilis, Bacillus licheniformis, Bacteroides fragilis,Clostridia perfringens, Clostridia difficile, Caulobacter crescentus,Lactococcus lactis, Methylobacterium extorquens, Neisseria meningirulls,Neisseria meningitidis, Pseudomonas fluorescens and Salmonellatyphimurium; and eukaryotic cells including, without limitation, yeaststrains, such as, e.g., those derived from Pichia pastoris, Pichiamethanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomycescerevisiae and Yarrowia lipolytica; insect cells and cell lines derivedfrom insects, such as, e.g., those derived from Spodoptera frugiperda,Trichoplusia ni, Drosophila melanogaster and Manduca sexta; andmammalian cells and cell lines derived from mammalian cells, such as,e.g., those derived from mouse, rat, hamster, porcine, bovine, equine,primate and human. Cell lines may be obtained from the American TypeCulture Collection, European Collection of Cell Cultures and the GermanCollection of Microorganisms and Cell Cultures. Non-limiting examples ofspecific protocols for selecting, making and using an appropriate cellline are described in e.g., INSECT CELL CULTURE ENGINEERING (Mattheus F.A. Goosen et al. eds., Marcel Dekker, 1993); INSECT CELL CULTURES:FUNDAMENTAL AND APPLIED ASPECTS (J. M. Vlak et al. eds., Kluwer AcademicPublishers, 1996); Maureen A. Harrison & Ian F. Rae, GENERAL TECHNIQUESOF CELL CULTURE (Cambridge University Press, 1997); CELL AND TISSUECULTURE: LABORATORY PROCEDURES (Alan Doyle et al eds., John Wiley andSons, 1998); R. Ian Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASICTECHNIQUE (Wiley-Liss, 4^(th) ed. 2000); ANIMAL CELL CULTURE: APRACTICAL APPROACH (John R. W. Masters ed., Oxford University Press,3^(rd) ed. 2000); MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001);BASIC CELL CULTURE: A PRACTICAL APPROACH (John M. Davis, Oxford Press,2^(nd) ed. 2002); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra,(2004). These protocols are routine procedures within the scope of oneskilled in the art and from the teaching herein.

The methods disclosed in the present specification include, in part,introducing into a cell a polynucleotide molecule. A polynucleotidemolecule introduced into a cell can be transiently or stably maintainedby that cell. Stably-maintained polynucleotide molecules may beextra-chromosomal and replicate autonomously, or they may be integratedinto the chromosomal material of the cell and replicatenon-autonomously. It is envisioned that any and all methods forintroducing a polynucleotide molecule disclosed in the presentspecification into a cell can be used. Methods useful for introducing anucleic acid molecule into a cell include, without limitation,chemical-mediated transfection such as, e.g., calciumphosphate-mediated, diethyl-aminoethyl (DEAE) dextran-mediated,lipid-mediated, polyethyleneimine (PEI)-mediated, polylysine-mediatedand polybrene-mediated; physical-mediated transfection, such as, e.g.,biolistic particle delivery, microinjection, protoplast fusion andelectroporation; and viral-mediated transfection, such as, e.g.,retroviral-mediated transfection, see, e.g., Introducing Cloned Genesinto Cultured Mammalian Cells, pp. 16.1-16.62 (Sambrook & Russell, eds.,Molecular Cloning A Laboratory Manual, Vol. 3, 3^(rd) ed. 2001). Oneskilled in the art understands that selection of a specific method tointroduce an expression construct into a cell will depend, in part, onwhether the cell will transiently contain an expression construct orwhether the cell will stably contain an expression construct. Theseprotocols are routine procedures within the scope of one skilled in theart and from the teaching herein.

In an aspect of this embodiment, a chemical-mediated method, termedtransfection, is used to introduce a polynucleotide molecule encoding amultivalent Clostridial toxin into a cell. In chemical-mediated methodsof transfection the chemical reagent forms a complex with the nucleicacid that facilitates its uptake into the cells. Such chemical reagentsinclude, without limitation, calcium phosphate-mediated, see, e.g.,Martin Jordan & Florian Worm, Transfection of adherent and suspendedcells by calcium phosphate, 33(2) Methods 136-143 (2004);diethyl-aminoethyl (DEAE) dextran-mediated, lipid-mediated, cationicpolymer-mediated like polyethyleneimine (PEI)-mediated andpolylysine-mediated and polybrene-mediated, see, e.g., Chun Zhang etal., Polyethylenimine strategies for plasmid delivery to brain-derivedcells, 33(2) Methods 144-150 (2004). Such chemical-mediated deliverysystems can be prepared by standard methods and are commerciallyavailable, see, e.g., CellPhect Transfection Kit (Amersham Biosciences,Piscataway, N.J.); Mammalian Transfection Kit, Calcium phosphate andDEAE Dextran, (Stratagene, Inc., La Jolla, Calif.); Lipofectamine™Transfection Reagent (Invitrogen, Inc., Carlsbad, Calif.); ExGen 500Transfection kit (Fermentas, Inc., Hanover, Md.), and SuperFect andEffectene Transfection Kits (Qiagen, Inc., Valencia, Calif.).

In another aspect of this embodiment, a physical-mediated method is usedto introduce a polynucleotide molecule encoding a multivalentClostridial toxin into a cell. Physical techniques include, withoutlimitation, electroporation, biolistic and microinjection. Biolisticsand microinjection techniques perforate the cell wall in order tointroduce the nucleic acid molecule into the cell, see, e.g., Jeike E.Biewenga et al., Plasmid-mediated gene transfer in neurons using thebiolistics technique, 71(1) J. Neurosci. Methods. 67-75 (1997); and JohnO'Brien & Sarah C. R. Lummis, Biolistic and diolistic transfection:using the gene gun to deliver DNA and lipophilic dyes into mammaliancells, 33(2) Methods 121-125 (2004). Electroporation, also termedelectropermeabilization, uses brief, high-voltage, electrical pulses tocreate transient pores in the membrane through which the nucleic acidmolecules enter and can be used effectively for stable and transienttransfections of all cell types, see, e.g., M. Golzio et al., In vitroand in vivo electric field-mediated permeabilization, gene transfer, andexpression, 33(2) Methods 126-135 (2004); and Oliver Gresch et al., Newnon-viral method for gene transfer into primary cells, 33(2) Methods151-163 (2004).

In another aspect of this embodiment, a viral-mediated method, termedtransduction, is used to introduce a polynucleotide molecule encoding amultivalent Clostridial toxin into a cell. In viral-mediated methods oftransient transduction, the process by which viral particles infect andreplicate in a host cell has been manipulated in order to use thismechanism to introduce a nucleic acid molecule into the cell.Viral-mediated methods have been developed from a wide variety ofviruses including, without limitation, retroviruses, adenoviruses,adeno-associated viruses, herpes simplex viruses, picornaviruses,alphaviruses and baculoviruses, see, e.g., Armin Blesch, Lentiviral andMLV based retroviral vectors for ex vivo and in vivo gene transfer,33(2) Methods 164-172 (2004); and Maurizio Federico, From lentivirusesto lentivirus vectors, 229 Methods Mol. Biol. 3-15 (2003); E. M.Poeschla, Non-primate lentiviral vectors, 5(5) Curr. Opin. Mol. Ther.529-540 (2003); Karim Benihoud et al, Adenovirus vectors for genedelivery, 10(5) Curr. Opin. Biotechnol. 440-447 (1999); H. Bueler,Adeno-associated viral vectors for gene transfer and gene therapy,380(6) Biol. Chem. 613-622 (1999); Chooi M. Lai et al., Adenovirus andadeno-associated virus vectors, 21(12) DNA Cell Biol. 895-913 (2002);Edward A. Burton et al., Gene delivery using herpes simplex virusvectors, 21(12) DNA Cell Biol. 915-936 (2002); Paola Grandi et al.,Targeting HSV amplicon vectors, 33(2) Methods 179-186 (2004); IlyaFrolov et al., Alphavirus-based expression vectors: strategies andapplications, 93(21) Proc. Natl. Acad. Sci. U.S.A. 11371-11377 (1996);Markus U. Ehrengruber, Alphaviral gene transfer in neurobiology, 59(1)Brain Res. Bull. 13-22 (2002); Thomas A. Kost & J. Patrick Condreay,Recombinant baculoviruses as mammalian cell gene-delivery vectors, 20(4)Trends Biotechnol. 173-180 (2002); and A. Huser & C. Hofmann,Baculovirus vectors: novel mammalian cell gene-delivery vehicles andtheir applications, 3(1) Am. J. Pharmacogenomics 53-63 (2003).

Adenoviruses, which are non-enveloped, double-stranded DNA viruses, areoften selected for mammalian cell transduction because adenoviruseshandle relatively large polynucleotide molecules of about 36 kb, areproduced at high titer, and can efficiently infect a wide variety ofboth dividing and non-dividing cells, see, e.g., Wim T. J. M. C. Hermenset al., Transient gene transfer to neurons and glia: analysis ofadenoviral vector performance in the CNS and PNS, 71(1) J. Neurosci.Methods 85-98 (1997); and Hiroyuki Mizuguchi et al., Approaches forgenerating recombinant adenovirus vectors, 52(3) Adv. Drug Deliv. Rev.165-176 (2001). Transduction using adenoviral-based system do notsupport prolonged protein expression because the nucleic acid moleculeis carried from an episome in the cell nucleus, rather than beingintegrated into the host cell chromosome. Adenoviral vector systems andspecific protocols for how to use such vectors are disclosed in, e.g.,ViraPower™ Adenoviral Expression System (Invitrogen, Inc., Carlsbad,Calif.) and ViraPower™ Adenoviral Expression System Instruction Manual25-0543 version A, Invitrogen, Inc., (Jul. 15, 2002); and AdEasy™Adenoviral Vector System (Stratagene, Inc., La Jolla, Calif.) andAdEasy™ Adenoviral Vector System Instruction Manual 064004f, Stratagene,Inc.

Nucleic acid molecule delivery can also use single-stranded RNAretroviruses, such as, e.g., oncoretroviruses and lentiviruses.Retroviral-mediated transduction often produce transduction efficienciesclose to 100%, can easily control the proviral copy number by varyingthe multiplicity of infection (MOI), and can be used to eithertransiently or stably transduce cells, see, e.g., Tiziana Tonini et al.,Transient production of retroviral- and lentiviral-based vectors for thetransduction of Mammalian cells, 285 Methods Mol. Biol. 141-148 (2004);Armin Blesch, Lentiviral and MLV based retroviral vectors for ex vivoand in vivo gene transfer, 33(2) Methods 164-172 (2004); FélixRecillas-Targa, Gene transfer and expression in mammalian cell lines andtransgenic animals, 267 Methods Mol. Biol. 417-433 (2004); and RolandWolkowicz et al., Lentiviral vectors for the delivery of DNA intomammalian cells, 246 Methods Mol. Biol. 391-411 (2004). Retroviralparticles consist of an RNA genome packaged in a protein capsid,surrounded by a lipid envelope. The retrovirus infects a host cell byinjecting its RNA into the cytoplasm along with the reversetranscriptase enzyme. The RNA template is then reverse transcribed intoa linear, double stranded cDNA that replicates itself by integratinginto the host cell genome. Viral particles are spread both vertically(from parent cell to daughter cells via the provirus) as well ashorizontally (from cell to cell via virions). This replication strategyenables long-term persistent expression since the nucleic acid moleculesof interest are stably integrated into a chromosome of the host cell,thereby enabling long-term expression of the protein. For instance,animal studies have shown that lentiviral vectors injected into avariety of tissues produced sustained protein expression for more than 1year, see, e.g., Luigi Naldini et al., In vivo gene delivery and stabletransduction of non-dividing cells by a lentiviral vector, 272(5259)Science 263-267 (1996). The Oncoretroviruses-derived vector systems,such as, e.g., Moloney murine leukemia virus (MoMLV), are widely usedand infect many different non-dividing cells. Lentiviruses can alsoinfect many different cell types, including dividing and non-dividingcells and possess complex envelope proteins, which allows for highlyspecific cellular targeting.

Retroviral vectors and specific protocols for how to use such vectorsare disclosed in, e.g., U.S. patent Nos. Manfred Gossen & HermannBujard, Tight control of gene expression in eukaryotic cells bytetracycline-responsive promoters, U.S. Pat. No. 5,464,758 (Nov. 7,1995) and Hermann Bujard & Manfred Gossen, Methods for regulating geneexpression, U.S. Pat. No. 5,814,618 (Sep. 29, 1998) David S. Hogness,Polynucleotides encoding insect steroid hormone receptor polypeptidesand cells transformed with same, U.S. Pat. No. 5,514,578 (May 7, 1996)and David S. Hogness, Polynucleotide encoding insect ecdysone receptor,U.S. Pat. No. 6,245,531 (Jun. 12, 2001); Elisabetta Vegeto et al.,Progesterone receptor having C. terminal hormone binding domaintruncations, U.S. Pat. No. 5,364,791 (Nov. 15, 1994), Elisabetta Vegetoet al., Mutated steroid hormone receptors, methods for their use andmolecular switch for gene therapy, U.S. Pat. No. 5,874,534 (Feb. 23,1999) and Elisabetta Vegeto et al., Mutated steroid hormone receptors,methods for their use and molecular switch for gene therapy, U.S. Pat.No. 5,935,934 (Aug. 10, 1999). Furthermore, such viral delivery systemscan be prepared by standard methods and are commercially available, see,e.g., BD™ Tet-Off and Tet-On Gene Expression Systems (BDBiosciences-Clonetech, Palo Alto, Calif.) and BD™ Tet-Off and Tet-OnGene Expression Systems User Manual, PT3001-1, BD Biosciences Clonetech,(Mar. 14, 2003), GeneSwitch™ System (Invitrogen, Inc., Carlsbad, Calif.)and GeneSwitch™ System A Mifepristone-Regulated Expression System forMammalian Cells version D, 25-0313, Invitrogen, Inc., (Nov. 4, 2002);ViraPower™ Lentiviral Expression System (Invitrogen, Inc., Carlsbad,Calif.) and ViraPower™ Lentiviral Expression System Instruction Manual25-0501 version E, Invitrogen, Inc., (Dec. 8, 2003); and CompleteControl® Retroviral Inducible Mammalian Expression System (Stratagene,La Jolla, Calif.) and Complete Control® Retroviral Inducible MammalianExpression System Instruction Manual, 064005e.

The methods disclosed in the present specification include, in part,expressing a multivalent Clostridial toxin from a polynucleotidemolecule. It is envisioned that any of a variety of expression systemsmay be useful for expressing a multivalent Clostridial toxin from apolynucleotide molecule disclosed in the present specification,including, without limitation, cell-based systems and cell-freeexpression systems. Cell-based systems include, without limitation,viral expression systems, prokaryotic expression systems, yeastexpression systems, baculoviral expression systems, insect expressionsystems and mammalian expression systems. Cell-free systems include,without limitation, wheat germ extracts, rabbit reticulocyte extractsand E. coli extracts and generally are equivalent to the methoddisclosed herein. Expression of a polynucleotide molecule using anexpression system can include any of a variety of characteristicsincluding, without limitation, inducible expression, non-inducibleexpression, constitutive expression, viral-mediated expression,stably-integrated expression, and transient expression. Expressionsystems that include well-characterized vectors, reagents, conditionsand cells are well-established and are readily available from commercialvendors that include, without limitation, Ambion, Inc. Austin Tex.; BDBiosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, SanDiego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; QIAGEN, Inc.,Valencia, Calif.; Roche Applied Science, Indianapolis, Ind.; andStratagene, La Jolla, Calif. Non-limiting examples on the selection anduse of appropriate heterologous expression systems are described ine.g., PROTEIN EXPRESSION. A PRACTICAL APPROACH(S. J. Higgins and B.David Hames eds., Oxford University Press, 1999); Joseph M. Fernandez &James P. Hoeffler, GENE EXPRESSION SYSTEMS. USING NATURE FOR THE ART OFEXPRESSION (Academic Press, 1999); and Meena Rai & Harish Padh,Expression Systems for Production of Heterologous Proteins, 80(9)CURRENT SCIENCE 1121-1128, (2001). These protocols are routineprocedures well within the scope of one skilled in the art and from theteaching herein.

A variety of cell-based expression procedures are useful for expressinga multivalent Clostridial toxin encoded by polynucleotide moleculedisclosed in the present specification. Examples included, withoutlimitation, viral expression systems, prokaryotic expression systems,yeast expression systems, baculoviral expression systems, insectexpression systems and mammalian expression systems. Viral expressionsystems include, without limitation, the ViraPower™ Lentiviral(Invitrogen, Inc., Carlsbad, Calif.), the Adenoviral Expression Systems(Invitrogen, Inc., Carlsbad, Calif.), the AdEasy™ XL Adenoviral VectorSystem (Stratagene, La Jolla, Calif.) and the ViraPort® Retroviral GeneExpression System (Stratagene, La Jolla, Calif.). Non-limiting examplesof prokaryotic expression systems include the Champion™ pET ExpressionSystem (EMD Biosciences-Novagen, Madison, Wis.), the TriEx™ BacterialExpression System (EMD Biosciences-Novagen, Madison, Wis.), theQIAexpress® Expression System (QIAGEN, Inc.), and the Affinity® ProteinExpression and Purification System (Stratagene, La Jolla, Calif.). Yeastexpression systems include, without limitation, the EasySelect™ PichiaExpression Kit (Invitrogen, Inc., Carlsbad, Calif.), the YES-Echo™Expression Vector Kits (Invitrogen, Inc., Carlsbad, Calif.) and theSpECTRA™ S. pombe Expression System (Invitrogen, Inc., Carlsbad,Calif.). Non-limiting examples of baculoviral expression systems includethe BaculoDirect™ (Invitrogen, Inc., Carlsbad, Calif.), the Bac-to-Bac®(Invitrogen, Inc., Carlsbad, Calif.), and the BD BaculoGold™ (BDBiosciences-Pharmigen, San Diego, Calif.). Insect expression systemsinclude, without limitation, the Drosophila Expression System (DES®)(Invitrogen, Inc., Carlsbad, Calif.), InsectSelect™ System (Invitrogen,Inc., Carlsbad, Calif.) and InsectDirect™ System (EMDBiosciences-Novagen, Madison, Wis.). Non-limiting examples of mammalianexpression systems include the T-REx™ (Tetracycline-RegulatedExpression) System (Invitrogen, Inc., Carlsbad, Calif.), the Flp-In™T-REx™ System (Invitrogen, Inc., Carlsbad, Calif.), the pcDNA™ system(Invitrogen, Inc., Carlsbad, Calif.), the pSecTag2 system (Invitrogen,Inc., Carlsbad, Calif.), the Exchanger® System, InterPlay™ Mammalian TAPSystem (Stratagene, La Jolla, Calif.), Complete Control® InducibleMammalian Expression System (Stratagene, La Jolla, Calif.) andLacSwitch® II Inducible Mammalian Expression System (Stratagene, LaJolla, Calif.).

Another procedure of expressing a multivalent Clostridial toxin encodedby polynucleotide molecule disclosed in the present specificationemploys a cell-free expression system such as, without limitation,prokaryotic extracts and eukaryotic extracts. Non-limiting examples ofprokaryotic cell extracts include the RTS 100 E. coli HY Kit (RocheApplied Science, Indianapolis, Ind.), the ActivePro In Vitro TranslationKit (Ambion, Inc., Austin, Tex.), the EcoPro™ System (EMDBiosciences-Novagen, Madison, Wis.) and the Expressway™ Plus ExpressionSystem (Invitrogen, Inc., Carlsbad, Calif.). Eukaryotic cell extractinclude, without limitation, the RTS 100 Wheat Germ CECF Kit (RocheApplied Science, Indianapolis, Ind.), the TnT® Coupled Wheat GermExtract Systems (Promega Corp., Madison, Wis.), the Wheat Germ IVT™ Kit(Ambion, Inc., Austin, Tex.), the Retic Lysate IVT™ Kit (Ambion, Inc.,Austin, Tex.), the PROTEINscript® II System (Ambion, Inc., Austin, Tex.)and the TnT® Coupled Reticulocyte Lysate Systems (Promega Corp.,Madison, Wis.).

Aspects of the present invention can also be described as follows:

-   1. A polypeptide comprising an endopeptidase domain; a translocation    domain effective to facilitate the movement of said endopeptidase    domain across an endosomal membrane; and at least two binding    domains, wherein: a first binding domain comprises a first ligand    binding, under physiological conditions, to a first cell surface    receptor displayed by the target cell; and a second binding domain    comprises a second ligand binding, under physiological conditions,    to a second cell surface receptor displayed by the target cell;    wherein said target cell internalizes said polypeptide upon binding    of said polypeptide to said target cell.-   2. The polypeptide of 1 wherein said second cell surface receptor    comprises a caveolin-binding domain.-   3. The polypeptide of 1 wherein said second cell surface receptor    comprises TNFR-1.-   4. The polypeptide of 3 wherein said second ligand comprises a    TNFR-1 binding protein selected from the group consisting of TNF-α    and a TNF-α derivative comprising a TNFR-1-binding domain.-   5. The polypeptide of 2 wherein said second ligand binds a glycosyl    phosphatylinositol (GPI)-linked membrane protein.-   6. The polypeptide of 1 wherein said second cell surface receptor    comprises an antibody variable (V) domain region.-   7. The polypeptide of 6 wherein said second ligand comprises an    antigen that selectively binds the antibody variable domain region    of said second cell surface receptor.-   8. The polypeptide of 1 wherein said second cell surface receptor    directly binds clatherin.-   9. The polypeptide of 1 wherein said second cell surface receptor    indirectly binds clatherin.-   10. The polypeptide of 9 wherein the second cell surface receptor    has the amino acid sequence tyrosine-X-arginine-phenylalanine near    the carboxy terminus.-   11. The polypeptide of 9 wherein the second cell surface receptor    binds an adaptin.-   12. The polypeptide of claim 11 wherein the second cell surface    receptor binds Adaptor Protein-2.-   13. The polypeptide of 1 wherein the first ligand selectively binds    a Clostridial neurotoxin-binding cell surface receptor.-   14. The polypeptide of 13 wherein the first ligand comprises a    Clostridial neurotoxin binding domain.-   15. The polypeptide of 15 wherein the first ligand comprises a    Clostridial neurotoxin binding domain selected from the group    consisting of a TeNT receptor binding domain, a BoNT-A receptor    binding domain, a BoNT-B receptor binding domain, a BoNT-C receptor    binding domain, a BoNT-C1 receptor binding domain, a BoNT-D receptor    binding domain, a BoNT-E receptor binding domain, a BoNT-F receptor    binding domain, a BoNT-G receptor binding domain HA or NTNH    β-trefoil domain of a neurotoxin associated protein (NAP) associated    with any Clostridial neurotoxin, and variants and isoforms of any of    the above.-   16. The polypeptide of 1 wherein first or second ligand comprises a    receptor-binding domain selected from the group consisting of: a    nerve growth factor (NGF) receptor binding domain; a leukemia    inhibitory factor (LIF) receptor binding domain; a basic fibroblast    growth factor (bFGF) receptor binding domain; a brain-derived    neurotrophic factor (BDNF) receptor binding domain; a neurotrophin-3    (NT-3) receptor binding domain; a hydra head activator peptide    (HHAP) receptor binding domain; a transforming growth factor 1    (TGF-1) receptor binding domain; a transforming growth factor 2    (TGF-2) receptor binding domain; a transforming growth factor 3    (TGF-3) receptor binding domain; an epidermal growth factor (EGF)    receptor binding domain; a ciliary neurotrophic factor (CNTF)    receptor binding domain; a tumor necrosis factor (TNF-) receptor    binding domain; an interleukin-1 (IL-1) receptor binding domain; an    interleukin-1 (IL-1) receptor binding domain; an interleukin-8    (IL-8) receptor binding domain; a bradykinin receptor binding    domain; a dynorphin receptor binding domain; a β-endorphin receptor    binding domain; an etorphine receptor binding domain; an    endomorphin-1 receptor binding domain; an endomorphin-2 receptor    binding domain; a leu-enkephalin receptor binding domain; a    met-enkephalin receptor binding domain; a galanin receptor binding    domain; a lofentanil receptor binding domain; a nociceptin receptor    binding domain; an antigen-binding variable region of an antibody    against the lactoseries carbohydrate epitopes found on the surface    of dorsal root ganglion neurons; an antigen-binding variable region    of an antibody binding any of the receptors for the binding domains    given above and an antibody against the surface expressed antigen    Thyl.-   17. The polypeptide of 16 wherein said first and second cell surface    receptors are identical.-   18. The polypeptide of 17 wherein the first and second ligands are    identical.-   19. The polypeptide of 16 wherein said first and second ligands are    different.-   20. The polypeptide of 15 wherein said second ligand comprises a    receptor-binding domain selected from the group consisting of: a    nerve growth factor (NGF) receptor binding domain; a leukemia    inhibitory factor (LIF) receptor binding domain; a basic fibroblast    growth factor (bFGF) receptor binding domain; a brain-derived    neurotrophic factor (BDNF) receptor binding domain; a neurotrophin-3    (NT-3) receptor binding domain; a hydra head activator peptide    (HHAP) receptor binding domain; a transforming growth factor 1    (TGF-1) receptor binding domain; a transforming growth factor 2    (TGF-2) receptor binding domain; a transforming growth factor 3    (TGF-3) receptor binding domain; an epidermal growth factor (EGF)    receptor binding domain; a ciliary neurotrophic factor (CNTF)    receptor binding domain; a tumor necrosis factor (TNF-) receptor    binding domain; an interleukin-1 (IL-1) receptor binding domain; an    interleukin-1 (IL-1) receptor binding domain; an interleukin-8    (IL-8) receptor binding domain; a bradykinin receptor binding    domain; a dynorphin receptor binding domain; a β-endorphin receptor    binding domain; an etorphine receptor binding domain; an    endomorphin-1 receptor binding domain; an endomorphin-2 receptor    binding domain; a leu-enkephalin receptor binding domain; a    met-enkephalin receptor binding domain; a galanin receptor binding    domain; a lofentanil receptor binding domain; a nociceptin receptor    binding domain; an antigen-binding variable region of an antibody    against the lactoseries carbohydrate epitopes found on the surface    of dorsal root ganglion neurons; an antigen-binding variable region    of an antibody binding any of the receptors for the binding domains    given above and an antibody against the surface expressed antigen    Thyl.-   21. The polypeptide of 16 wherein said second cell surface receptor    comprises a caveolin-binding domain.-   22. The polypeptide of 16 wherein said second cell surface receptor    comprises TNFR-1.-   23. The polypeptide of 22 wherein said second ligand comprises a    TNFR-1 binding protein selected from the group consisting of TNF-α    and a TNF-α derivative comprising a TNFR-1-binding domain.-   24. The polypeptide of 21 wherein said second ligand binds a    glycosyl phosphatylinositol (GPI)-linked membrane protein.-   25. The polypeptide of 16 wherein said second cell surface receptor    comprises an antibody variable (V) domain region.-   26. The polypeptide of 25 wherein said second ligand comprises an    antigen that selectively binds the antibody variable domain region    of said second cell surface receptor.-   27. The polypeptide of 16 wherein said second cell surface receptor    directly or indirectly binds clatherin.-   28. The polypeptide of 27 wherein said second cell surface receptor    indirectly binds clatherin.-   29. The polypeptide of 27 wherein the second cell surface receptor    has the amino acid sequence tyrosine-X-arginine-phenylalanine near    the carboxy terminus.-   30. The polypeptide of 27 wherein the second cell surface receptor    directly binds an adaptin.-   31. The polypeptide of 30 wherein the second cell surface receptor    binds Adaptor Protein-2.-   32. A method for inhibiting or decreasing exocytosis or secretion in    a target cell comprising contacting said target cell under    physiological conditions with an effective dose of a multivalent    Clostridial toxin composition comprising: an endopeptidase domain; a    translocation domain effective to facilitate the movement of said    endopeptidase domain from an endocytotic vesicle to the cytoplasm of    said target cell; and at least two binding domains, wherein: a first    binding domain comprises a first ligand selectively binding under    physiological conditions to a first cell surface receptor    selectively displayed by the target cell; and a second binding    domain comprises a second ligand binding, under physiological    conditions, to a second cell surface receptor displayed by the    target cell; wherein said endopeptidase domain catalyzes cleavage of    a intracellular SNARE protein within the target cell, thereby    inhibiting or decreasing exocytosis or secretion by said cell.-   33. The method of 32 wherein said second cell surface receptor    comprises a caveolin-binding domain.-   34. The method of 32 wherein said second cell surface receptor    comprises TNFR-1.-   35. The method of 34 wherein said second ligand comprises a TNFR-1    binding protein selected from the group consisting of TNF-α and a    TNF-α derivative comprising a TNFR-1-binding domain.-   36. The method of 33 wherein said second ligand binds a glycosyl    phosphatylinositol (GPI)-linked membrane protein.-   37. The method of 32 wherein said second cell surface receptor    comprises an antibody variable (V) domain region.-   38. The method of 37 wherein said second ligand comprises an antigen    that selectively binds the antibody variable domain region of said    second cell surface receptor.-   39. The method of 32 wherein said second cell surface receptor    directly binds clatherin.-   40. The method of 32 wherein said second cell surface receptor    indirectly binds clatherin.-   41. The method of 39 wherein the second cell surface receptor has    the amino acid sequence tyrosine-X-arginine-phenylalanine near the    carboxy terminus.-   42. The method of 39 wherein the second cell surface receptor binds    an adaptin.-   43. The method of 42 wherein the second cell surface receptor binds    Adaptor Protein-2.-   44. The method of 32 wherein the first ligand selectively binds a    Clostridial neurotoxin-binding cell surface receptor.-   45. The method of 44 wherein the first ligand comprises a    clostridial neurotoxin binding domain.-   46. The method of 45 wherein the first ligand comprises a    clostridial neurotoxin binding domain selected from the group    consisting of a TeNT receptor binding domain, a BoNT-A receptor    binding domain, a BoNT-B receptor binding domain, a BoNT-C receptor    binding domain, a BoNT-C1 receptor binding domain, a BoNT-D receptor    binding domain, a BoNT-E receptor binding domain, a BoNT-F receptor    binding domain, a BoNT-G receptor binding domain HA or NTNH β    trefoil domain of a neurotoxin associated protein (NAP) associated-   47. The method of 32 wherein first or second ligand comprises a    receptor-binding domain selected from the group consisting of: a    nerve growth factor (NGF) receptor binding domain; a leukemia    inhibitory factor (LIF) receptor binding domain; a basic fibroblast    growth factor (bFGF) receptor binding domain; a brain-derived    neurotrophic factor (BDNF) receptor binding domain; a neurotrophin-3    (NT-3) receptor binding domain; a hydra head activator peptide    (HHAP) receptor binding domain; a transforming growth factor 1    (TGF-1) receptor binding domain; a transforming growth factor 2    (TGF-2) receptor binding domain; a transforming growth factor 3    (TGF-3) receptor binding domain; an epidermal growth factor (EGF)    receptor binding domain; a ciliary neurotrophic factor (CNTF)    receptor binding domain; a tumor necrosis factor (TNF-) receptor    binding domain; an interleukin-1 (IL-1) receptor binding domain; an    interleukin-1 (IL-1) receptor binding domain; an interleukin-8    (IL-8) receptor binding domain; a bradykinin receptor binding    domain; a dynorphin receptor binding domain; a β-endorphin receptor    binding domain; an etorphine receptor binding domain; an    endomorphin-1 receptor binding domain; an endomorphin-2 receptor    binding domain; a leu-enkephalin receptor binding domain; a    met-enkephalin receptor binding domain; a galanin receptor binding    domain; a lofentanil receptor binding domain; a nociceptin receptor    binding domain; an antigen-binding variable region of an antibody    against the lactoseries carbohydrate epitopes found on the surface    of dorsal root ganglion neurons; an antigen-binding variable region    of an antibody binding any of the receptors for the binding domains    given above and an antibody against the surface expressed antigen    Thyl.-   48. The method of 47 wherein said first and second cell surface    receptors are identical.-   49. The method of 48 wherein the first and second ligands are    identical.-   50. The method of 47 wherein said first and second ligands are    different.-   51. The method of 46 wherein said second ligand comprises a    receptor-binding domain selected from the group consisting of: a    nerve growth factor (NGF) receptor binding domain; a leukemia    inhibitory factor (LIF) receptor binding domain; a basic fibroblast    growth factor (bFGF) receptor binding domain; a brain-derived    neurotrophic factor (BDNF) receptor binding domain; a neurotrophin-3    (NT-3) receptor binding domain; a hydra head activator peptide    (HHAP) receptor binding domain; a transforming growth factor 1    (TGF-1) receptor binding domain; a transforming growth factor 2    (TGF-2) receptor binding domain; a transforming growth factor 3    (TGF-3) receptor binding domain; an epidermal growth factor (EGF)    receptor binding domain; a ciliary neurotrophic factor (CNTF)    receptor binding domain; a tumor necrosis factor (TNF-) receptor    binding domain; an interleukin-1 (IL-1) receptor binding domain; an    interleukin-1 (IL-1) receptor binding domain; an interleukin-8    (IL-8) receptor binding domain; a bradykinin receptor binding    domain; a dynorphin receptor binding domain; β-endorphin receptor    binding domain; an etorphine receptor binding domain; an    endomorphin-1 receptor binding domain; an endomorphin-2 receptor    binding domain; a leu-enkephalin receptor binding domain; a    met-enkephalin receptor binding domain; a galanin receptor binding    domain; a lofentanil receptor binding domain; a nociceptin receptor    binding domain; an antigen-binding variable region of an antibody    against the lactoseries carbohydrate epitopes found on the surface    of dorsal root ganglion neurons; an antigen-binding variable region    of an antibody binding any of the receptors for the binding domains    given above and an antibody against the surface expressed antigen    Thyl.-   52. The method of 47 wherein said second cell surface receptor    comprises a caveolin-binding domain.-   53. The method of 46 wherein said second cell surface receptor    comprises TNFR-1.-   54. The method of 53 wherein said second ligand comprises a TNFR-1    binding protein selected from the group consisting of TNF-□ and a    TNF-□ derivative comprising a TNFR-1-binding domain.-   55. The method of 52 wherein said second ligand binds a glycosyl    phosphatylinositol (GPI)-linked membrane protein.-   56. The method of 16 wherein said second cell surface receptor    comprises an antibody variable (V) domain region.-   57. The method of 56 wherein said second ligand comprises an antigen    that selectively binds the antibody variable domain region of said    second cell surface receptor.-   58. The method of 47 wherein said second cell surface receptor    directly or indirectly binds clatherin.-   59. The method of 58 wherein said second cell surface receptor    indirectly binds clatherin.-   60. The method of 58 wherein the second cell surface receptor has    the amino acid sequence tyrosine-X-arginine-phenylalanine near the    carboxy terminus.-   61. The method of 58 wherein the second cell surface receptor    directly binds an adaptin.-   62. The method of 61 wherein the second cell surface receptor binds    Adaptor Protein-2.-   63. The method of 32 wherein said exocytosis or secretion is    associated with a pathological condition related to a condition    selected from the group consisting of a movement disorder, a    disorder of the sensory nervous system, acute or chronic pain,    cancer, pancreatitis, hyperhydrosis, glandular disorders, viral    infections, and cystic fibrosis.-   64. The method of 32 wherein the multivalent Clostridial toxin is    administered by injection.-   65. A nucleic acid molecule encoding a polypeptide according to any    one of 1-31.

Aspects of the present invention can also be described as follows:

-   1. A multivalent Clostridial toxin comprising:    -   a) a Clostridial toxin enzymatic domain capable of executing an        enzymatic target modification step of a Clostridial toxin        intoxication process;    -   b) a Clostridial toxin translocation domain capable of executing        a translocation step of a Clostridial toxin intoxication        process;    -   c) a first binding domain capable of executing a cell binding        step of a Clostridial toxin intoxication process by selectively        binding a first cell surface receptor displayed by the target        cell; and    -   d) a second binding domain capable of executing a cell binding        step of a Clostridial toxin intoxication process by selectively        binding a second cell surface receptor displayed by the target        cell; and    -   e) a protease cleavage site, wherein cleavage of the protease        cleavage site converts the single-chain form of the modified        Clostridial toxin into the di-chain form.-   2. The multivalent Clostridial toxin according to 1, wherein the    multivalent Clostridial toxin comprises amino to carboxyl linear    organization comprising a binding domain 1, a translocation domain,    a binding domain 2, a protease cleavage site and an enzymatic    domain.-   3. The multivalent Clostridial toxin according to 1, wherein the    multivalent Clostridial toxin comprises an amino to carboxyl linear    organization comprising an enzymatic domain, a protease cleavage    site, a binding domain 1, a translocation domain and a binding    domain 2-   4. The multivalent Clostridial toxin according to 1, wherein the    multivalent Clostridial toxin comprises an amino to carboxyl linear    organization comprising an enzymatic domain, a protease cleavage    site, a translocation domain, a binding domain 1 and a binding    domain 2-   5. The multivalent Clostridial toxin according to 1, wherein the    multivalent Clostridial toxin comprises an amino to carboxyl linear    organization comprising an enzymatic domain, a protease cleavage    site, a translocation domain, a binding domain 2 and a binding    domain 1-   6. The multivalent Clostridial toxin according to 1, wherein the    multivalent Clostridial toxin comprises an amino to carboxyl linear    organization comprising a binding domain 1, an enzymatic domain, a    protease cleavage site, a translocation domain and a binding domain    2-   7. The multivalent Clostridial toxin according to 1, wherein the    multivalent Clostridial toxin comprises amino to carboxyl linear    organization comprising a translocation domain, a binding domain 2,    a protease cleavage site, a binding domain 1 and an enzymatic domain-   8. The multivalent Clostridial toxin according to 1, wherein the    multivalent Clostridial toxin comprises an amino to carboxyl linear    organization comprising a translocation domain, a binding domain 2,    a protease cleavage site, an enzymatic domain and a binding domain 1-   9. The multivalent Clostridial toxin according to 1, wherein the    multivalent Clostridial toxin comprises an amino to carboxyl linear    organization comprising a binding domain 2, a translocation domain,    a protease cleavage site, a binding domain 1 and an enzymatic domain-   10. The multivalent Clostridial toxin according to 1, wherein the    protease cleavage site is an endogenous protease cleavage site or an    exogenous protease cleavage site.-   11. The multivalent Clostridial toxin according to 10, wherein the    endogenous protease cleavage site is selected from the group    consisting of a BoNT/A di-chain loop protease cleavage site, a    BoNT/B di-chain loop protease cleavage site, a BoNT/C1 di-chain loop    protease cleavage site, a BoNT/D di-chain loop protease cleavage    site, a BoNT/E di-chain loop protease cleavage site, a BoNT/F    di-chain loop protease cleavage site, a BoNT/G di-chain loop    protease cleavage site and a TeNT di-chain loop protease cleavage    site.-   12. The multivalent Clostridial toxin according to 10, wherein the    exogenous protease cleavage site is selected from the group    consisting of a bovine enterokinase protease cleavage site, a    Tobacco Etch Virus protease cleavage site, a Human Rhinovirus 3C    protease cleavage site, a SUMO/ULP-1 protease cleavage site, a    Thrombin protease cleavage site, and a Factor Xa protease cleavage    site.-   13. A multivalent Clostridial toxin comprising:    -   a) a Clostridial toxin enzymatic domain capable of executing an        enzymatic target modification step of a Clostridial toxin        intoxication process;    -   b) a Clostridial toxin translocation domain capable of executing        a translocation step of a Clostridial toxin intoxication        process;    -   c) a first binding domain capable of executing a cell binding        step of a Clostridial toxin intoxication process by selectively        binding a first cell surface receptor displayed by the target        cell;    -   d) a second binding domain capable of executing a cell binding        step of a Clostridial toxin intoxication process by selectively        binding a second cell surface receptor displayed by the target        cell;    -   e) a first protease cleavage site, wherein cleavage of the first        protease cleavage site converts the single-chain form of the        modified Clostridial toxin into the di-chain form; and    -   f) a second protease cleavage site; wherein cleavage of the        second protease causes a structural confirmation in the first        and second binding domains which facilitates the selectively        binding activity of the first binding domain, the second binding        domain, or both the first and second binding domains.-   14. The multivalent Clostridial toxin according to 13, wherein the    multivalent Clostridial toxin comprises an amino to carboxyl linear    organization comprising an enzymatic domain, a first protease    cleavage site, a translocation domain, a binding domain 1, a second    protease cleavage site and a binding domain 2-   15. The multivalent Clostridial toxin according to 13, wherein the    multivalent Clostridial toxin comprises an amino to carboxyl linear    organization comprising an enzymatic domain, a first protease    cleavage site, a translocation domain, a binding domain 2, a second    protease cleavage site and a binding domain 1-   16. The modified Clostridial toxin according to 13, wherein the    first protease cleavage site is an endogenous protease cleavage site    or an exogenous protease cleavage site.-   17. The multivalent Clostridial toxin according to 16, wherein the    endogenous protease cleavage site is selected from the group    consisting of a BoNT/A di-chain loop protease cleavage site, a    BoNT/B di-chain loop protease cleavage site, a BoNT/C1 di-chain loop    protease cleavage site, a BoNT/D di-chain loop protease cleavage    site, a BoNT/E di-chain loop protease cleavage site, a BoNT/F    di-chain loop protease cleavage site, a BoNT/G di-chain loop    protease cleavage site and a TeNT di-chain loop protease cleavage    site.-   18. The multivalent Clostridial toxin according to 16, wherein the    exogenous protease cleavage site is selected from the group    consisting of a bovine enterokinase protease cleavage site, a    Tobacco Etch Virus protease cleavage site, a Human Rhinovirus 3C    protease cleavage site, a SUMO/ULP-1 protease cleavage site, a    Thrombin protease cleavage site, and a Factor Xa protease cleavage    site.-   19. The modified Clostridial toxin according to 13, wherein the    second protease cleavage site is an endogenous protease cleavage    site or an exogenous protease cleavage site.-   20. The multivalent Clostridial toxin according to 19, wherein the    endogenous protease cleavage site is selected from the group    consisting of a BoNT/A di-chain loop protease cleavage site, a    BoNT/B di-chain loop protease cleavage site, a BoNT/C1 di-chain loop    protease cleavage site, a BoNT/D di-chain loop protease cleavage    site, a BoNT/E di-chain loop protease cleavage site, a BoNT/F    di-chain loop protease cleavage site, a BoNT/G di-chain loop    protease cleavage site and a TeNT di-chain loop protease cleavage    site.-   21. The multivalent Clostridial toxin according to 19, wherein the    exogenous protease cleavage site is selected from the group    consisting of a bovine enterokinase protease cleavage site, a    Tobacco Etch Virus protease cleavage site, a Human Rhinovirus 3C    protease cleavage site, a SUMO/ULP-1 protease cleavage site, a    Thrombin protease cleavage site, and a Factor Xa protease cleavage    site.-   22. The multivalent Clostridial toxin according to either 1 or 13,    wherein the Clostridial toxin enzymatic domain is selected from the    group consisting of a BoNT/A enzymatic domain, a BoNT/B enzymatic    domain, a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a    BoNT/E enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G    enzymatic domain, a TeNT enzymatic domain, a BaNT enzymatic domain    and a BuNT enzymatic domain.-   23. The multivalent Clostridial toxin according to 22, wherein the    Clostridial toxin enzymatic domain comprises an amino acid sequence    selected from the group consisting of amino acids 1-448 of SEQ ID    NO: 1, amino acids 1-441 SEQ ID NO: 2, amino acids 1-449 of SEQ ID    NO: 3, amino acids 1-445 of SEQ ID NO: 4, amino acids 1-422 of SEQ    ID NO: 5, amino acids 1-439 of SEQ ID NO: 6, amino acids 1-446 of    SEQ ID NO: 7, amino acid 1-457 of SEQ ID NO: 8, amino acid 1-431 of    SEQ ID NO: 9, and amino acid 1-422 of SEQ ID NO: 10.-   24. The multivalent Clostridial toxin according either 1 or 13,    wherein the Clostridial toxin translocation domain is selected from    the group consisting of a BoNT/A translocation domain, a BoNT/B    translocation domain, a BoNT/C1 translocation domain, a BoNT/D    translocation domain, a BoNT/E translocation domain, a BoNT/F    translocation domain, a BoNT/G translocation domain, a TeNT    translocation domain, a BaNT translocation domain and a BuNT    translocation domain.-   25. The multivalent Clostridial toxin according to 24, wherein the    Clostridial toxin translocation domain comprises an amino acid    sequence selected from the group consisting of amino acids 449-873    of SEQ ID NO: 1, amino acids 442-860 SEQ ID NO: 2, amino acids    450-868 of SEQ ID NO: 3, amino acids 446-864 of SEQ ID NO: 4, amino    acids 423-847 of SEQ ID NO: 5, amino acids 440-866 of SEQ ID NO: 6,    amino acids 447-865 of SEQ ID NO: 7, amino acid 458-881 of SEQ ID    NO: 8, amino acid 432-857 of SEQ ID NO: 9, and amino acid 423-847 of    SEQ ID NO: 10.-   26. The multivalent Clostridial toxin according either 1 or 13,    wherein the first binding domain comprises a binding domain selected    from the group consisting of a Clostridial toxin binding domain, a    Clostridial non-toxin associated protein β-trefoil domain and an FGF    β-trefoil domain.-   27. The multivalent Clostridial toxin according to 26, wherein the    Clostridial toxin binding domain is selected from the group    consisting of a BoNT/A binding domain, a BoNT/B binding domain, a    BoNT/C1 binding domain, a BoNT/D binding domain, a BoNT/E binding    domain, a BoNT/F binding domain, a BoNT/G binding domain, a TeNT    binding domain, a BaNT binding domain, and a BuNT binding domain.-   28. The multivalent Clostridial toxin according to 26, wherein the    Clostridial toxin binding domain comprises a BoNT/A H_(C) binding    domain, a BoNT/B H_(C) binding domain, a BoNT/C1 H_(C) binding    domain, a BoNT/D H_(C) binding domain, a BoNT/E H_(C) binding    domain, a BoNT/F H_(C) binding domain, a BoNT/G H_(C) binding    domain, a TeNT H_(C) binding domain, a BaNT H_(C) binding domain,    and a BuNT H_(C) binding domain.-   29. The multivalent Clostridial toxin according to 28, wherein the    Clostridial toxin binding domain comprises an amino acid sequence    selected from the group consisting of amino acids 874-1296 of SEQ ID    NO: 1, amino acids 861-1291 SEQ ID NO: 2, amino acids 869-1291 of    SEQ ID NO: 3, amino acids 865-1276 of SEQ ID NO: 4, amino acids    848-1252 of SEQ ID NO: 5, amino acids 867-1274 of SEQ ID NO: 6,    amino acids 866-1297 of SEQ ID NO: 7, amino acid 882-1315 of SEQ ID    NO: 8, amino acid 858-1268 of SEQ ID NO: 9, and amino acid 848-1251    of SEQ ID NO: 10.-   30. The multivalent Clostridial toxin according to 26, wherein the    Clostridial toxin binding domain comprises a BoNT/A H_(CC) binding    domain, a BoNT/B H_(CC) binding domain, a BoNT/C1 H_(CC) binding    domain, a BoNT/D H_(CC) binding domain, a BoNT/E H_(CC) binding    domain, a BoNT/F H_(CC) binding domain, a BoNT/G H_(CC) binding    domain, a TeNT H_(CC) binding domain, a BaNT H_(CC) binding domain,    and a BuNT H_(CC) binding domain.-   31. The multivalent Clostridial toxin according to 30, wherein the    Clostridial toxin binding domain comprises an amino acid sequence    selected from the group consisting of amino acids 874-1110 of SEQ ID    NO: 1, amino acids 861-1097 SEQ ID NO: 2, amino acids 869-1111 of    SEQ ID NO: 3, amino acids 865-1098 of SEQ ID NO: 4, amino acids    848-1085 of SEQ ID NO: 5, amino acids 867-1105 of SEQ ID NO: 6,    amino acids 866-1105 of SEQ ID NO: 7, amino acid 882-1127 of SEQ ID    NO: 8, amino acid 858-1094 of SEQ ID NO: 9, and amino acid 848-1085    of SEQ ID NO: 10.-   32. The multivalent Clostridial toxin according to 26, wherein the    Clostridial non-toxin associated protein β-trefoil domain is    selected from the group consisting of a BoNT/A HA-33 β-trefoil    domain, a BoNT/B HA-33 β-trefoil domain, a BoNT/C1 HA-33 β-trefoil    domain, a BoNT/D HA-33 β-trefoil domain, a BoNT/A HA-17 β-trefoil    domain, a BoNT/B HA-17 β-trefoil domain, a BoNT/C1 HA-17 β-trefoil    domain, a BoNT/D HA-17 β-trefoil domain, a BoNT/A NTNH β-trefoil    domain, a BoNT/B NTNH β-trefoil domain, a BoNT/C1 NTNH β-trefoil    domain, a BoNT/D NTNH β-trefoil domain, a BoNT/E NTNH β-trefoil    domain, a BoNT/F NTNH β-trefoil domain, AND a BoNT/G NTNH β-trefoil    domain.-   33. The multivalent Clostridial toxin according to 32, wherein the    BoNT/A HA-33 β-trefoil domain comprises amino acids 10-144 of SEQ ID    NO: 11, amino acids 151-293 of SEQ ID NO: 11, amino acids 10-144 or    amino acids 151-293 of SEQ ID NO: 12, amino acids 10-144 of SEQ ID    NO: 13, amino acids 151-293 of SEQ ID NO: 13, amino acids 10-146 of    SEQ ID NO: 14, amino acids 153-294 of SEQ ID NO: 14, amino acids    10-144 of SEQ ID NO: 15, or amino acids 151-279 of SEQ ID NO: 15.-   34. The multivalent Clostridial toxin according to 32, wherein the    BoNT/B HA-33 β-trefoil domain comprises amino acids 10-144 of SEQ ID    NO: 16, amino acids 151-292 of SEQ ID NO: 16, amino acids 10-146 of    SEQ ID NO: 17, or amino acids 153-291 of SEQ ID NO: 17.-   35. The multivalent Clostridial toxin according to 32, wherein the    BoNT/C1 HA-33 β-trefoil domain comprises amino acids 10-141 of SEQ    ID NO: 18, amino acids 148-285 of SEQ ID NO: 18, amino acids 10-141    of SEQ ID NO: 19, or amino acids 148-286 of SEQ ID NO: 19.-   36. The multivalent Clostridial toxin according to 32, wherein the    BoNT/D HA-33 β-trefoil domain comprises amino acids 10-141 of SEQ ID    NO: 20, or amino acids 148-286 of SEQ ID NO: 20.-   37. The multivalent Clostridial toxin according to 32, wherein the    BoNT/A HA-17 β-trefoil domain comprises amino acids 9-146 of SEQ ID    NO: 21.-   38. The multivalent Clostridial toxin according to 32, wherein the    BoNT/B HA-17 β-trefoil domain comprises amino acids 9-146 of SEQ ID    NO: 22.-   39. The multivalent Clostridial toxin according to 32, wherein the    BoNT/C1 HA-17 β-trefoil domain comprises amino acids 9-146 of SEQ ID    NO: 23.-   40. The multivalent Clostridial toxin according to 32, wherein the    BoNT/D HA-17 β-trefoil domain comprises amino acids 9-146 of SEQ ID    NO: 24.-   41. The multivalent Clostridial toxin according to 32, wherein the    BoNT/A NTNH β-trefoil domain comprises amino acids 1050-1194 of SEQ    ID NO: 25, amino acids 1050-1199 of SEQ ID NO: 26, or amino acids    1050-1194 of SEQ ID NO: 27.-   42. The multivalent Clostridial toxin according to 32, wherein the    BoNT/B NTNH β-trefoil domain comprises amino acids 1049-1198 of SEQ    ID NO: 28.-   43. The multivalent Clostridial toxin according to 32, wherein the    BoNT/C1 NTNH β-trefoil domain comprises amino acids 1049-1197 of SEQ    ID NO:29.-   44. The multivalent Clostridial toxin according to 32, wherein the    BoNT/D NTNH β-trefoil domain comprises amino acids 1049-1197 of SEQ    ID NO: 30.-   45. The multivalent Clostridial toxin according to 32, wherein the    BoNT/E NTNH β-trefoil domain comprises amino acids 1014-1163 of SEQ    ID NO: 31.-   46. The multivalent Clostridial toxin according to 32, wherein the    BoNT/F NTNH β-trefoil domain comprises amino acids 1016-1160 of SEQ    ID NO: 32, or amino acids 1017-1166 of SEQ ID NO: 33.-   47. The multivalent Clostridial toxin according to 32, wherein the    BoNT/G NTNH β-trefoil domain comprises amino acids 1050-1197 of SEQ    ID NO: 34.-   48. The multivalent Clostridial toxin according to 26, wherein the    FGF β-trefoil domain is selected from the group consisting of a    FGF-1 β-trefoil domain, a FGF-2 β-trefoil domain, a FGF-4 β-trefoil    domain, a FGF-8 β-trefoil domain, a FGF-9 β-trefoil domain, a FGF-17    β-trefoil domain and a FGF-18 β-trefoil domain.-   49. The multivalent Clostridial toxin according to 26, wherein the    FGF β-trefoil domain is selected from the group consisting of amino    acids 26-155 of SEQ ID NO: 35, amino acids 29-155 of SEQ ID NO: 36,    amino acids 83-206 of SEQ ID NO: 37, amino acids 43-172 of SEQ ID    NO: 38, amino acids 63-196 of SEQ ID NO: 39, amino acids 55-183 of    SEQ ID NO: 40 and amino acids 54-183 of SEQ ID NO: 41.-   50. The multivalent Clostridial toxin according either 1 or 13,    wherein the first binding domain comprises a binding domain selected    from the group consisting of a glucagon like hormone, a    neurohormone, a neuroregulatory cytokine, a neurotrophin, a growth    factor, and an axon guidance signaling molecule.-   51. The multivalent Clostridial toxin according to 50, wherein the    glucagon like hormone is selected from the group consisting of a    GRPP, a glucagon-like peptide-1 (GLP-1), a glucagon-like peptide-2    (GLP-2), a glucagon, an oxyntomodulin (OXY), pituitary adenylate    cyclase activating peptide (PACAP), GHRH, vasoactive intestinal    peptide-1 (VIP-1), vasoactive intestinal peptide-2 (VIP-2), gastric    inhibitory polypeptide (GIP), secretin, gastrin, GRP, Ghrelin (GHS),    glicentin, glicentin-related polypeptide (GRPP), and a    calcitonin-related peptidesvisceral gut peptide.-   52. The multivalent Clostridial toxin according to 50, wherein the    glucagon like hormone comprises amino acids 21-50 of SEQ ID NO: 42,    amino acids 53-81 of SEQ ID NO: 42, amino acids 53-89 of SEQ ID NO:    42, amino acids 98-124 of SEQ ID NO: 42, amino acids 146-178 of SEQ    ID NO: 42, amino acids 132-158 of SEQ ID NO: 43, amino acids 32-58,    of SEQ ID NO: 44, amino acids 32-75 of SEQ ID NO: 44, amino acids    81-107 of SEQ ID NO: 45, amino acids 125-151 of SEQ ID NO: 45, amino    acids 81-107 of SEQ ID NO: 46, amino acids 124-150 of SEQ ID NO: 46,    amino acids 52-78 of SEQ ID NO: 47, amino acids 52-93 of SEQ ID NO:    47, amino acids 28-54 of SEQ ID NO: 48, amino acids 76-92 of SEQ ID    NO: 49, amino acids 59-92 of SEQ ID NO: 49, amino acids 41-50 of SEQ    ID NO: 50, amino acids 24-50 of SEQ ID NO: 50, or amino acids 99-112    of SEQ ID NO: 51.-   53. The multivalent Clostridial toxin according to 50, wherein the    neurohormone is selected from the group consisting of    corticotropin-releasing hormone (CCRH) and parathyroid hormone    (PTH).-   54. The multivalent Clostridial toxin according to 50, wherein the    neurohormone comprises a amino acids 159-193 of SEQ ID NO: 52, amino    acids 154-194 of SEQ ID NO: 52, amino acids 35-70 of SEQ ID NO: 53    and amino acids 145-177 of SEQ ID NO: 53.-   55. The multivalent Clostridial toxin according to 50, wherein the    neuroregulatory cytokine is selected from the group consisting of    ciliary neurotrophic factor (CNTF), glycophorin-A (GPA), leukemia    inhibitory factor (LIF), an interleukin (IL), onostatin M (OSM),    cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC),    neuroleukin (NL), VEGF, insulin-like growth factor-1 (IGF-1),    insulin-like growth factor-2 (IGF-2) and epidermal growth factor    (EGF).-   56. The multivalent Clostridial toxin according to 50, wherein the    neurohormone comprises SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56,    SEQ ID NO: 57, SEQ ID NO: 58, amino acids 123-265 of SEQ ID NO: 59,    amino acids 21-153 of SEQ ID NO: 60, amino acids 57-210 of SEQ ID    NO: 61, amino acids 21-99 of SEQ ID NO: 62, amino acids 31-94 of SEQ    ID NO: 62, amino acids 37-173 of SEQ ID NO: 63, amino acids 19-178    of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, amino acids 52-109    of SEQ ID NO: 66, amino acids 49-118 of SEQ ID NO: 66, amino acids    31-84 of SEQ ID NO: 67, amino acids 25-180 of SEQ ID NO: 67 or SEQ    ID NO: 68.-   57. The multivalent Clostridial toxin according to 50, wherein the    neurotrophin is selected from the group consisting of nerve growth    factor (NGF), brain-derived growth factor (BDNF), neurotrophin-3    (NT-3) and neurotrophin-4/5 (NT-4/5).-   58. The multivalent Clostridial toxin according to 50, wherein the    neurotrophin comprises amino acids 139-257 of SEQ ID NO: 69, amino    acids 133-240 of SEQ ID NO: 70, amino acids 129-247 of SEQ ID NO:    70, amino acids 144-249 of SEQ ID NO: 71, amino acids 19-257 of SEQ    ID NO: 71, amino acids 89-202 of SEQ ID NO: 72 and amino acids    81-210 of SEQ ID NO: 72.-   59. The multivalent Clostridial toxin according to 50, wherein the    growth factor is selected from the group consisting of glial cell    derived neurotrophic factor (GDNF), neurturin (NRTN), persephrin    (PSPN), artemin (ARTN), TGFβ1, TGFβ2, TGFβ3, TGFβ4, BMP2, BMP3,    BMP4, BMP5, BMP6, BMP7, BMP8, BMP10, GDF1, GDF2, GDF3, GDF5, GDF6,    GDF7, GDF8, GDF10, GDF11, GDF15, activin A, activin B, activin C,    activin E and inhibin A.-   60. The multivalent Clostridial toxin according to 50, wherein the    growth factor selected from the group consisting of amino acids    118-211 of SEQ ID NO: 73, amino acids 107-196 of SEQ ID NO: 74,    amino acids 96-197 of SEQ ID NO: 74, amino acids 66-155 of SEQ ID    NO: 75, amino acids 123-218 of SEQ ID NO: 76, amino acids 293-390 of    SEQ ID NO: 77, amino acids 317-414 of SEQ ID NO: 78, amino acids    315-412 of SEQ ID NO: 79, amino acids 276-373 of SEQ ID NO: 80,    amino acids 296-396 of SEQ ID NO: 81, amino acids 370-472 of SEQ ID    NO: 82, amino acids 309-409 of SEQ ID NO: 83, amino acids 353-454 of    SEQ ID NO: 84, amino acids 323-454 of SEQ ID NO: 84, amino acids    412-513 of SEQ ID NO: 85, amino acids 374-513 of SEQ ID NO: 85,    amino acids 330-431 of SEQ ID NO: 86, amino acids 293-431 of SEQ ID    NO: 86, amino acids 301-402 of SEQ ID NO: 87, amino acids 323-424 of    SEQ ID NO: 88, amino acids 267-372 of SEQ ID NO: 89, amino acids    327-429 of SEQ ID NO: 90, amino acids 264-364 of SEQ ID NO: 91,    amino acids 400-501 of SEQ ID NO: 92, amino acids 354-455 of SEQ ID    NO: 93, amino acids 352-450 of SEQ ID NO: 94, amino acids 281-375 of    SEQ ID NO: 95; amino acids 376-478 of SEQ ID NO: 96; amino acids    313-407 of SEQ ID NO: 97; amino acids 211-308 of SEQ ID NO: 98;    amino acids 321-426 of SEQ ID NO: 99; amino acids 303-406 of SEQ ID    NO: 100; amino acids 247-352 of SEQ ID NO: 101; amino acids 237-352    of SEQ ID NO: 101; amino acids 247-350 of SEQ ID NO: 102; amino    acids 262-366 of SEQ ID NO: 103; or amino acids 233-366 of SEQ ID    NO: 103.-   61. The multivalent Clostridial toxin according either 1 or 13,    wherein the first binding domain comprises a binding domain selected    from the group consisting of an opioid peptide, a melanocortin    peptide, a galanin peptide, a granin peptide, a tachykinin peptide,    a cholecystokinin peptide, a Neuropeptide Y related peptide, a kinin    peptide, a protease activated receptor (PAR) peptide, a somatostatin    peptide, a leukemia inhibitor factor peptide, and an interleukin-1    peptide.-   62. The multivalent Clostridial toxin according to 61, wherein the    opioid peptide is selected from the group consisting of an    enkephalin peptide, a BAM22 peptide, an endomorphin peptide, an    endorphin peptide, a dynorphin peptide, a nociceptin peptide and a    hemorphin peptide.-   63. The multivalent Clostridial toxin according to 62, wherein the    enkephalin peptide is selected from the group consisting of a    Leu-enkephalin, a Met-enkephalin, a Met-enkephalin MRGL and a    Met-enkephalin MRF-   64. The multivalent Clostridial toxin according to 62, wherein the    enkephalin peptide comprises SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID    NO: 106 or SEQ ID NO: 107.-   65. The multivalent Clostridial toxin according to 62, wherein the    BAM22 peptide is selected from the group consisting of a BAM22    peptide (1-12), a BAM22 peptide (6-22), a BAM22 peptide (8-22) or a    BAM22 peptide (1-22).-   66. The multivalent Clostridial toxin according to 62, wherein the    BAM22 peptide comprises amino acids 1-12 of SEQ ID NO: 108, amino    acids 6-22 of SEQ ID NO: 108, amino acids 8-22 of SEQ ID NO: 108,    amino acids 1-22 of SEQ ID NO: 108, amino acids 1-12 of SEQ ID NO:    109, amino acids 6-22 of SEQ ID NO: 109, amino acids 8-22 of SEQ ID    NO: 109, amino acids 1-22 of SEQ ID NO: 109, amino acids 1-12 of SEQ    ID NO: 110, amino acids 6-22 of SEQ ID NO: 110, amino acids 8-22 of    SEQ ID NO: 110, amino acids 1-22 of SEQ ID NO: 110, amino acids 1-12    of SEQ ID NO: 111, amino acids 6-22 of SEQ ID NO: 111, amino acids    8-22 of SEQ ID NO: 111, amino acids 1-22 of SEQ ID NO: 111, amino    acids 1-12 of SEQ ID NO: 112, amino acids 6-22 of SEQ ID NO: 112,    amino acids 8-22 of SEQ ID NO: 112, amino acids 1-22 of SEQ ID NO:    112, amino acids 1-12 of SEQ ID NO: 113, amino acids 6-22 of SEQ ID    NO: 113, amino acids 8-22 of SEQ ID NO: 113, or amino acids 1-22 of    SEQ ID NO: 113.-   67. The multivalent Clostridial toxin according to 62, wherein the    endomorphin peptide is selected from the group consisting of an    endomorphin-1 and an endomorphin-2.-   68. The multivalent Clostridial toxin according to 62, wherein the    endomorphin peptide comprises SEQ ID NO: 114, or SEQ ID NO: 115.-   69. The multivalent Clostridial toxin according to 62, wherein the    endorphin peptide is selected from the group consisting of an    endorphin-α, a neoendorphin-α, an endorphin-β, a neoendorphin-β or    an endorphin-γ.-   70. The multivalent Clostridial toxin according to 62, wherein the    altered targeting domain comprises an endorphin selected from the    group consisting of SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117,    SEQ ID NO: 118, SEQ ID NO: 119, or SEQ ID NO: 120.-   71. The multivalent Clostridial toxin according to 62, wherein the    dynorphin peptide is selected from the group consisting of a    dynorphin A, a dynorphin B and a rimorphin.-   72. The multivalent Clostridial toxin according to 62, wherein the    dynorphin peptide comprises SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID    NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO:    127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131,    SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ    ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID    NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO:    144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148,    SEQ ID NO: 149, SEQ ID NO: 150, or SEQ ID NO: 151.-   73. The multivalent Clostridial toxin according to 62, wherein the    nociceptin peptide selected from the group consisting of a    nociceptin RK, a nociceptin, a neuropeptide 1, a neuropeptide 2 or a    neuropeptide 3.-   74. The multivalent Clostridial toxin according to 62, wherein the    nociceptin peptide comprises SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID    NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO:    158, SEQ ID NO: 159, SEQ ID NO: 160, or SEQ ID NO: 161.-   75. The multivalent Clostridial toxin according to 61, wherein the    melanocortin peptide is selected from the group consisting of an    α-melanocyte stimulating hormones (α-MSH), α-melanocyte stimulating    hormones (β-MSH), a γ-melanocyte stimulating hormones (γ-MSH), an    adrenocorticotropin (ACTH), a Corticotropin-like intermediary    peptide (CLIP), a β-lipotropin (β-LPH) and a γ-lipotropin (γ-LPH).-   76. The multivalent Clostridial toxin according to 61, wherein the    melanocortin peptide is selected from the group consisting SEQ ID    NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO:    166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170,    or SEQ ID NO: 171.-   77. The multivalent Clostridial toxin according to 61, wherein the    galanin peptide is selected from the group consisting of a galanin    and a galanin message-associated peptide (GMAP).-   78. The multivalent Clostridial toxin according to 61, wherein the    galanin peptide comprises SEQ ID NO: 172, or SEQ ID NO: 173.-   79. The multivalent Clostridial toxin according to 61, wherein the    grainin peptide is selected from the group consisting of a    chromogranin A peptide, a chromogranin B peptide and a chromogranin    C peptide.-   80. The multivalent Clostridial toxin according to 79, wherein the    chromogranin A peptide is selected from the group consisting of a    β-granin, a vasostatin, a chromostatin, a pancreastatin, a WE-14, a    catestatin, a parastatin and a GE-25.-   81. The modified Clostridial toxin according to 79, wherein the    chromogranin A peptide comprises SEQ ID NO: 174, SEQ ID NO: 175, SEQ    ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID    NO: 180, or SEQ ID NO: 181.-   82. The multivalent Clostridial toxin according to 79, wherein the    chromogranin B peptide is selected from the group consisting of a    GAWK peptide, an adrenomedullary peptide and a secretolytin.-   83. The multivalent Clostridial toxin according to 79, wherein the    chromogranin B peptide comprises SEQ ID NO: 182, SEQ ID NO: 183, SEQ    ID NO: 184, SEQ ID NO: 185, or SEQ ID NO: 186-   84. The multivalent Clostridial toxin according to 79, wherein the    chromogranin C peptide is selected from the group consisting of a    secretoneurin, a EM66 and a manserin.-   85. The multivalent Clostridial toxin according to 79, wherein the    chromogranin C peptide comprises SEQ ID NO: 187.-   86. The multivalent Clostridial toxin according to 61, wherein the    tachykinin peptide is selected from the group consisting of a    Substance P, a neuropeptide K (NPK), a neuropeptide gamma, a    neurokinin A, a neurokinin B, a hemokinin and a endokinin.-   87. The multivalent Clostridial toxin according to 61, wherein the    tachykinin peptide comprises SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID    NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO:    194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198    and SEQ ID NO: 199.-   88. The multivalent Clostridial toxin according to 61, wherein the    cholecystokinin peptide is selected from the group consisting of a    cholecystokinin 58, a cholecystokinin 39, a cholecystokinin 33, a    cholecystokinin 12 and a cholecystokinin 8.-   89. The multivalent Clostridial toxin according to 88, wherein the    cholecystokinin 58 is selected from the group consisting of SEQ ID    NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO:    204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208,    SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ    ID NO: 213, SEQ ID NO: 214, or SEQ ID NO: 215.-   90. The multivalent Clostridial toxin according to 88, wherein the    cholecystokinin 39 is selected from the group consisting of amino    acids 20-58 of SEQ ID NO: 200, amino acids 20-58 of SEQ ID NO: 201,    amino acids 20-58 of SEQ ID NO: 202, amino acids 20-58 of SEQ ID NO:    203, amino acids 20-58 of SEQ ID NO: 204, amino acids 20-58 of SEQ    ID NO: 205, amino acids 20-58 of SEQ ID NO: 207, amino acids 20-58    of SEQ ID NO: 208, amino acids 20-58 of SEQ ID NO: 209, amino acids    20-58 of SEQ ID NO: 210, amino acids 20-58 of SEQ ID NO: 211, amino    acids 20-58 of SEQ ID NO: 212, amino acids 20-58 of SEQ ID NO: 213,    amino acids 20-58 of SEQ ID NO: 214, or amino acids 20-58 of SEQ ID    NO: 215.-   91. The multivalent Clostridial toxin according to 88, wherein the    cholecystokinin 33 is selected from the group consisting of amino    acids 26-58 of SEQ ID NO: 200, amino acids 26-58 of SEQ ID NO: 201,    amino acids 26-58 of SEQ ID NO: 202, amino acids 26-58 of SEQ ID NO:    203, amino acids 26-58 of SEQ ID NO: 204, amino acids 26-58 of SEQ    ID NO: 205, amino acids 26-58 of SEQ ID NO: 207, amino acids 26-58    of SEQ ID NO: 208, amino acids 26-58 of SEQ ID NO: 209, amino acids    26-58 of SEQ ID NO: 210, amino acids 26-58 of SEQ ID NO: 211, amino    acids 26-58 of SEQ ID NO: 212, amino acids 26-58 of SEQ ID NO: 213,    amino acids 26-58 of SEQ ID NO: 214 or amino acids 26-58 of SEQ ID    NO: 215.-   92. The multivalent Clostridial toxin according to 88, wherein the    cholecystokinin 12 is selected from the group consisting of amino    acids 47-58 of SEQ ID NO: 200, amino acids 47-58 of SEQ ID NO: 210    or amino acids 47-58 of SEQ ID NO: 214.-   93. The multivalent Clostridial toxin according to 88, wherein the    cholecystokinin 8 is amino acids 51-58 of SEQ ID NO: 200.-   94. The multivalent Clostridial toxin according to 61, wherein the    Neuropeptide Y related peptide is selected from the group consisting    of a Neuropeptide Y (NPY), a Peptide YY (PYY), a Pancreatic peptide    (PP) and a Pancreatic icosapeptide (PIP).-   95. The multivalent Clostridial toxin according to 61, wherein the    Neuropeptide Y related peptide comprises SEQ ID NO: 216, SEQ ID NO:    217, SEQ ID NO: 218, SEQ ID NO: 219, or SEQ ID NO: 220.-   96. The multivalent Clostridial toxin according to 61, wherein the    kinin peptide is selected from the group consisting of a bradykinin,    a kallidin, a desArg⁹ bradykinin and a desArg¹⁰ bradykinin.-   97. The multivalent Clostridial toxin according to 61, wherein the    kinin peptide comprises SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO:    229, or SEQ ID NO: 230.-   98. The multivalent Clostridial toxin according to 61, wherein the    PAR peptide is selected from the group consisting of a PAR1 peptide,    a PAR2 peptide, a PAR3 peptide and a PAR4 peptide.-   99. The modified Clostridial toxin according to 61, wherein the PAR    peptide comprises amino acids 42-47, amino acids 42-55, amino acids    29-64 or amino acids 1-64 of SEQ ID NO: 231; amino acids 35-40,    amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID    NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60 or    amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids    48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234.-   100. The multivalent Clostridial toxin according either 1 or 13,    wherein the first binding domain comprises a binding domain selected    from the group consisting of a PTD.-   101. The multivalent Clostridial toxin according to 100, wherein the    PTD is selected from the group consisting of a herpes simplex virus    type 1 VP22 protein translocating sequence, a SV-40 virus large T    translocating sequence, a TAT translocating sequence, an adenovirus    translocating sequence, a synthetic integrin binding domain    translocating sequence, a Kaposi fibroblast growth factor membrane    translocating sequence, a nuclear localization signal, a Transportan    translocating sequence, a ciliary neurotrophic factor translocating    sequence, a caveolin, an interleukin 1-β translocating sequence, a    thioredoxin translocating sequence, a fibroblast growth factor-1    translocating sequence, a fibroblast growth factor-2 translocating    sequence, an integrin β1 translocating sequence, an integrin β3    translocating sequence, a lactoferrin translocating sequence, a    homeodomain translocating sequence, like, a penetratin translocating    sequence, an Engrailed-1 translocating sequence, an Engrailed-2    translocating sequence, a Hoxa-5 translocating sequence, a Hoxb-4    translocating sequence, and a Hoxc-8 translocating sequence.-   102. The multivalent Clostridial toxin according to 100, wherein the    PTD comprises SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID    NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO:    242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246,    SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ    ID NO: 251, SEQ ID NO: 252, or SEQ ID NO: 253.-   103. A polynucleotide molecule encoding a multivalent Clostridial    toxin according to any one of Claims 1-102.-   104. The polynucleotide molecule according to Claims 103, wherein    the polynucleotide molecule is an expression construct.-   105. A method of producing a multivalent Clostridial toxin    comprising the step of expressing in a cell a polynucleotide    according to 52-   106. A method of producing a multivalent Clostridial toxin    comprising the steps of    -   a) introducing in a cell an expression construct comprising a        polynucleotide according to 52; and    -   b) expressing the expression construct in the cell.

EXAMPLES Example 1 Construction of a Multivalent Clostridial NeurotoxinComprising Two Clostridial Toxin Binding Domains

This example illustrates how to make a multivalent Clostridial toxincomprising two modified Clostridial toxin binding domains with enhancedbinding activity using site-directed mutagenesis.

A polynucleotide molecule encoding BoNT/A (SEQ ID NO: 1) and furthercomprising a polynucleotide sequence comprising a repeat of amino acids874-1296 of the binding domain of BoNT/A at the amino terminus issynthesized using standard procedures (BlueHeron® Biotechnology,Bothell, Wash.). The basic strategy is set forth in FIG. 5A.Oligonucleotides of 20 to 50 bases in length are synthesized usingstandard phosphoramidite synthesis. These oligonucleotides arehybridized into double stranded duplexes that are ligated together toassemble the full-length polynucleotide molecule. This polynucleotidemolecule is cloned using standard molecular biology methods into apUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/multivalentClostridial toxin(AA)1. The synthesized polynucleotide molecule isverified by sequencing using Big Dye Terminator™ Chemistry 3.1 (AppliedBiosystems, Foster City, Calif.) and an ABI 3100 sequencer (AppliedBiosystems, Foster City, Calif.).

If desired, an expression-optimized polynucleotide molecule encodingBoNT/A (SEQ ID NO: 1) can be synthesized in order to improve expressionin an Escherichia coli strain. The polynucleotide molecule encoding theBoNT/A can be modified to 1) contain synonymous codons typically presentin native polynucleotide molecules of an Escherichia coli strain; 2)contain a G+C content that more closely matches the average G+C contentof native polynucleotide molecules found in an Escherichia coli strain;3) reduce polymononucleotide regions found within the polynucleotidemolecule; and/or 4) eliminate internal regulatory or structural sitesfound within the polynucleotide molecule, see, e.g., Lance E. Steward etal. Optimizing Expression of Active Botulinum Toxin Type E, PCT PatentSerial No. 2005/020578 (Jun. 9, 2005); Lance E. Steward et al.Optimizing Expression of Active Botulinum Toxin Type A, PCT PatentSerial No. 2005/027917 (Aug. 3, 2005). Once sequence optimization iscomplete, oligonucleotides of 20 to 50 bases in length are synthesizedusing standard phosphoramidite synthesis. These oligonucleotides arehybridized into double stranded duplexes that are ligated together toassemble the full-length polynucleotide molecule. This polynucleotidemolecule is cloned using standard molecular biology methods into apUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/multivalentClostridial toxin1/(AA)1:ECopt. The synthesized polynucleotide moleculeis verified by sequencing using Big Dye Terminator™ Chemistry 3.1(Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer(Applied Biosystems, Foster City, Calif.). Is so desired, optimizationto a different organism, such as, e.g., a yeast strain, an insectcell-line or a mammalian cell line, can be done, see, e.g., Steward,supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); and Steward,supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005).

A similar cloning strategy is used to make pUCBHB1 cloning constructscomprising a polynucleotide molecule encoding BoNT/B of SEQ ID NO: 2; apolynucleotide molecule encoding BoNT/C1 of SEQ ID NO: 3; apolynucleotide molecule encoding BoNT/D of SEQ ID NO: 4; apolynucleotide molecule encoding BoNT/E of SEQ ID NO: 5; apolynucleotide molecule encoding BoNT/F of SEQ ID NO: 6; apolynucleotide molecule encoding BoNT/G of SEQ ID NO: 7; apolynucleotide molecule encoding TeNT of SEQ ID NO: 8; a polynucleotidemolecule encoding BaNT of SEQ ID NO: 9; a polynucleotide moleculeencoding BuNT of SEQ ID NO: 10; wherein the additional binding domaincan be selected from, e.g., any of the H_(C) or H_(CC) binding domainslisted in Table 1. In addition, one skilled in the art can modifyClostridial toxins, such as, e.g., to include an exogenous proteasecleavage site within the di-chain loop region, or flexible spacerregions. Likewise, a similar cloning strategy can be used to make otherdomain orientations as, e.g., as set forth in FIG. 4A, 4B, 4C, 4D, 5B,5C, 5D, 6A or 6B.

To construct pET29/multivalent Clostridial toxin/A(AA)1, apUCBHB1/multivalent Clostridial toxin(AA)1 construct is digested withrestriction endonucleases that 1) excise the insert comprising the openreading frame of SEQ ID NO: 1 (with the added nucleotide sequenceencoding the N terminal Binding 1 site) encoding the multivalentClostridial toxin(AA)1; and 2) enable this insert to be operably-linkedto a pET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insertis subcloned using a T4 DNA ligase procedure into a pET29 vector that isdigested with appropriate restriction endonucleases to yieldpET29/multivalent Clostridial toxin/A(AA)1. The ligation mixture istransformed into chemically competent E. coli DH5α cells (Invitrogen,Inc, Carlsbad, Calif.) using a heat shock method, plated on 1.5%Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of Kanamycin, andplaced in a 37° C. incubator for overnight growth. Bacteria containingexpression constructs are identified as kanamycin resistant colonies.Candidate constructs are isolated using an alkaline lysis plasmidmini-preparation procedure and analyzed by restriction endonucleasedigest mapping to determine the presence and orientation of the insert.This cloning strategy yields a pET29 expression construct comprising thepolynucleotide molecule encoding the BoNT/A of SEQ ID NO: 1operably-linked to an additional N-terminal binding site comprising thenative binding region of BoNT/A.

A similar cloning strategy is used to make pET29 expression constructscomprising polynucleotide molecule encoding the multivalent Clostridialtoxins discussed above comprising BoNT/B of SEQ ID NO: 2; apolynucleotide molecule encoding BoNT/C1 of SEQ ID NO: 3; apolynucleotide molecule encoding BoNT/D of SEQ ID NO: 4; apolynucleotide molecule encoding BoNT/E of SEQ ID NO: 5; apolynucleotide molecule encoding BoNT/F of SEQ ID NO: 6; apolynucleotide molecule encoding BoNT/G of SEQ ID NO: 7; apolynucleotide molecule encoding TeNT of SEQ ID NO: 8, a polynucleotidemolecule encoding BaNT of SEQ ID NO: 9 and a polynucleotide moleculeencoding BuNT of SEQ ID NO: 10, each with an additional binding site.

To construct a multivalent Clostridial toxin comprising one or moremodified binding domain with enhanced binding activity, specific aminoacids influencing binding activity will be changed. For example, it isalready known that amino acids Trp 1101, Gly 1102, Leu 1105, Tyr 1111,Tyr 1112, Gly 1158, Ile 1163, Asp 1179, Glu 1203, Phe 1252, Ser 1264,Trp 1266, Tyr 1267, Gln 1270, Gly 1279 and Trp 1282 of SEQ ID NO: 1(within the binding site) are important for function. To determine whichamino acid substitutions could enhance the binding activity of a BoNT/Abinding domain, computational protein design algorithms will generatenovel binding domains with optimized properties. The crystal structureof a BoNT/A binding domain will be used as the starting template forcomputational calculations. Potential amino acid candidates will beidentified using a combined output from Protein Design Automation®(PDA®) and Sequence Prediction Algorithm™ (SPA™) calculations. For PDAcalculations, the conformations of amino acids at variable positionswill be represented as a set of backbone-independent side chain rotamersderived from the rotamer library. The energies of all possiblecombinations of the considered amino acids at the chosen variablepositions will be calculated using a force field containing termsdescribing van der Waals, solvation, electrostatic, and hydrogen bondinteractions. The optimal (ground state) sequence will be determinedusing a Dead End Elimination (DEE) algorithm, and a Monte Carlo (MC)algorithm will be used to evaluate the energies of similar sequencesaround the predicted ground state. SPA calculations utilize a geneticalgorithm to screen for low energy sequences, with energies beingcalculated during each round of “evolution” for those sequences beingsampled. The conformations of amino acids will be represented as a setof side chain rotamers derived from a backbone-independent rotamerlibrary using a flexible rotamer model. SPA calculations will generatesequences which will be subsequently clustered computationally intogroups of similar sequences using a nearest neighbor single linkagehierarchical clustering algorithm. Critical contact amino acids will befixed in both sequence and conformation and calculations will be carriedout to evaluate single and combinatorial substitutions at variable aminoacids. All amino acids in contact with these residues will be floated,that is the amino acid conformation but not the amino acid identity willbe allowed to vary to allow for conformational adjustments. Finalexperimental substitutions will be chosen based on their predictedenergies relative to the naturally occurring BoNT/A binding domain andtheir occupancy, that is the number times the substitution occurred inthe set of 1000 MC or genetic algorithm sequences. Two sets of designcalculations will be carried out using Rosetta to identify substitutionspredicted to stabilize the BoNT/A binding domain. In the first round,only single amino acid substitutions will be modeled. In a second round,interface amino acids will be allowed to change to all 20 naturallyoccurring amino acids including the native amino acid type, butexcluding cysteine, simultaneously. In each case, amino acid side chainscontacting the substituted amino acid side chains will be repacked(allowing all rotamers of the native amino acid type). Sequences andconformations with low energies will be selected using a Monte-Carlosimulated annealing procedure. All resulting protein complex models willbe rescored by computing a predicted binding energy. Final sequences areselected for the lowest binding energy.

To use this information to generate one or more modified Clostridialneurotoxin binding domain, candidate amino acids identified as describedabove will be changed using site-directed in vitro mutagenesis. A 50 μLreaction will be assembled using pET29/multivalent Clostridialtoxin/A(AA)1 as a template, sense and antisense oligonucleotidesencoding the desired amino acid change identified above, and reagentsincluded with the QuickChange® II XL Site-Directed Mutagenesis kit(Stratagene, La Jolla, Calif.). The polymerase chain reaction (PCR) mixwill contain 5 μL of 10× Buffer, 1 μL of deoxyribonucleotides (dNTPs), 1μL of PfuUltra™ High Fidelity DNA polymerase (2.5 units/μL), 125 ng ofeach primer, 100 ng of template DNA, and nuclease-free water to a finalvolume of 50 μL. The thermocycler conditions will be: one cycle of 95°C. for 60 seconds; 16 cycles of 95° C. for 30 seconds, 55° C. for 60seconds, and 72° C. for 10 minutes; one cycle of 72° C. for 5 minutes;and 4° C. to hold. Following thermocycling, 1 μL of DpnI restrictionenzyme (Stratagene, La Jolla, Calif.) will be added to the reaction andwill be incubated for 1 hour at 37° C. to digest the template DNA. Thereaction will be purified by QIAquick kit (QIAGEN, Inc., Valencia,Calif.) and will be analysis by agarose gel electrophoresis to determinethat the reaction produced full-length plasmid. The mutagenesis productswill be transformed into chemically competent E. coli DH5α cells(Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on1.5% Luria-Bertani agar plates (pH 7.0) containing 100 μg/mL ofAmpicillin, and will be placed in a 37° C. incubator for overnightgrowth. Candidate mutagenesis constructs will be isolated as Ampicillinresistant colonies and will be analyzed using an alkaline lysis plasmidmini-preparation procedure to isolate the expression construct andrestriction endonuclease digests to determine the presence of theinsert. The incorporation of the point mutation will be determined bysequence analysis of candidate plasmid constructs.

To test the binding activity of multivalent Clostridial toxinscomprising binding domains derived from BoNT/A, the soluble portion ofFGFR3 will be expressed recombinantly for use in surface plasmonresonance (SPR) binding assays, e.g., Biacore® (Biacore Inc.,Piscataway, N.J.). The soluble portion of FGFR3 will be expressed as afusion to streptavidin and the receptor will then be immobilized on anappropriate sensor chip. Utilizing a Biacore® instrument, changes inlocal refractive index as a result of receptor binding will be measuredas a change in the SPR angle. The rates of change in the SPR angle willthen be analyzed to determine association rate (K_(on)), dissociationrate (K_(off)) and the dissociation equilibrium constant(K_(D)=K_(off)/K_(on)). Multivalent Clostridial neurotoxin derivativescomprising binding domains derived from BoNT/A exhibit either anincreased association rate, a decreased dissociation rate, both anincreased association rate and a decreased dissociation rate, or adecreased dissociation equilibrium constant relative to the measurementsobtained from the naturally occurring BoNT/A from which the multivalentClostridial toxin is derived.

The same methods and rationale may be used to make and test the affinityof any multivalent Clostridial toxin comprising additional bindingdomain derived from a Clostridial toxin. Generally, but not exclusively,a multivalent Clostridial toxin is tested relative to the Clostridialtoxin with which it shares the greatest homology, particularly in thebinding domains.

Example 2 Construction of a Multivalent Clostridial NeurotoxinComprising a Non-Toxin Associated Protein

A polynucleotide molecule encoding BoNT/A-Nterm33/A is synthesized usingstandard procedures (BlueHeron® Biotechnology, Bothell, Wash.), asdescribed in Example 1. BoNT/A-Nterm33/A is a BoNT/A modified to replaceamino acids in the second binding domain (the N-terminal binding regionof the multivalent Clostridial toxin described in Example 1)corresponding to amino acids 1111-1296 of SEQ ID NO: 1, a BoNT/Aβ-trefoil domain, with amino acids 151 to 293 of SEQ ID NO: 9, a HA-33β-trefoil domain from a Clostridial botulinum serotype A strain. Ifdesired, an expression optimized polynucleotide molecule encodingBoNT/A-Nterm33/A can be synthesized in order to improve expression in toa different organism, such as, e.g., an Escherichia coli strain, a yeaststrain, an insect cell-line or a mammalian cell line, can be done, see,e.g., Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005);and Steward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005).The synthesized polynucleotide molecule is verified by sequencing usingBig Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City,Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City,Calif.).

A similar cloning strategy is used to make pUCBHB1 cloning constructsfor multivalent Clostridial toxin BoNT/B-Nterm33/A, a multivalentClostridial toxin based on BoNT/B where amino acids 1098-1291 of SEQ IDNO: 2 are replaced with amino acids 151 to 293 of SEQ ID NO: 11;multivalent Clostridial toxin BoNT/C1-Nterm33/A, a multivalentClostridial toxin based on BoNT/C1 where amino acids 1112-1291 of SEQ IDNO: 3 are replaced with amino acids 151 to 293 of SEQ ID NO: 11;multivalent Clostridial toxin BoNT/D-Nterm33/A, a multivalentClostridial toxin based on BoNT/D where amino acids 1099-1276 of SEQ IDNO: 4 are replaced with amino acids 151 to 293 of SEQ ID NO: 11;multivalent Clostridial toxin BoNT/E-Nterm33/A, a multivalentClostridial toxin based on BoNT/E where amino acids 1086-1252 of SEQ IDNO: 5 are replaced with amino acids 151 to 293 of SEQ ID NO: 11;multivalent Clostridial toxin BoNT/F-Nterm33/A, a multivalentClostridial toxin based on BoNT/F where amino acids 1106-1274 of SEQ IDNO: 6 are replaced with amino acids 151 to 293 of SEQ ID NO: 11;multivalent Clostridial toxin BoNT/G-Nterm33/A, a multivalentClostridial toxin based on BoNT/G where amino acids 1106-1297 of SEQ IDNO: 7 are replaced with amino acids 151 to 293 of SEQ ID NO: 11; andmultivalent Clostridial toxin TeNT-Nterm33/A, a modified TeNT whereamino acids 1128-1315 of SEQ ID NO: 8 are replaced with amino acids 151to 293 of SEQ ID NO: 11.

Similarly, the β-trefoil domain from a Clostridial toxin indicated abovecan be replaced with a non-toxin associated protein β-trefoil domaincomprising amino acids 10-144 of SEQ ID NO: 11; amino acids 10-144 ofSEQ ID NO: 12; amino acids 10-144 of SEQ ID NO: 13; amino acids 10-146of SEQ ID NO: 14; amino acids 10-144 of SEQ ID NO: 15; amino acids10-144 of SEQ ID NO: 16; amino acids 10-146 of SEQ ID NO: 17; aminoacids 10-141 of SEQ ID NO: 18; amino acids 10-141 of SEQ ID NO: 19;amino acids 10-141 of SEQ ID NO: 20; amino acids 151-293 of SEQ ID NO:12; amino acids 151-293 of SEQ ID NO: 13; amino acids 153-294 of SEQ IDNO: 14; amino acids 151-279 of SEQ ID NO: 15; amino acids 151-292 of SEQID NO: 16; amino acids 153-291 of SEQ ID NO: 17; amino acids 148-285 ofSEQ ID NO: 18; amino acids 148-286 of SEQ ID NO: 19; amino acids 148-286of SEQ ID NO: 20; amino acids 9-146 of SEQ ID NO: 21; amino acids 9-146of SEQ ID NO: 22; amino acids 9-146 of SEQ ID NO: 23; amino acids 9-146of SEQ ID NO: 24; amino acids 1050-1194 of SEQ ID NO: 25; amino acids1050-1199 of SEQ ID NO: 26; amino acids 1050-1194 of SEQ ID NO: 27;amino acids 1049-1198 of SEQ ID NO: 28; amino acids 1049-1197 of SEQ IDNO: 29; amino acids 1049-1197 of SEQ ID NO: 30; amino acids 1014-1163 ofSEQ ID NO: 31; amino acids 1016-1160 of SEQ ID NO: 32; amino acids1017-1166 of SEQ ID NO: 33; and amino acids 1050-1199 of SEQ ID NO: 34.

To construct pET29/multivalent Clostridial toxin/BoNT/A-Nterm33/A, apUCBHB1/BoNT/A-33/A construct is digested with restriction endonucleasesthat 1) excise the insert comprising the open reading frame encoding theBoNT/A-Nterm33/A; and 2) enable this insert to be operably-linked to apET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert issubcloned using a T4 DNA ligase procedure into a pET29 vector that isdigested with appropriate restriction endonucleases to yieldpET29/BoNT/A-Nterm33/A. The ligation mixture is transformed intochemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad,Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agarplates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C.incubator for overnight growth. Bacteria containing expressionconstructs are identified as kanamycin resistant colonies. Candidateconstructs are isolated using an alkaline lysis plasmid mini-preparationprocedure and analyzed by restriction endonuclease digest mapping todetermine the presence and orientation of the insert. This cloningstrategy yielded a pET29 expression construct comprising thepolynucleotide molecule encoding a multivalent Clostridial toxinBoNT/A-Nterm33/A operably-linked to a carboxyl terminal polyhistidineaffinity binding peptide.

A similar cloning strategy is used to make pET29 expression constructscomprising a polynucleotide molecule encoding for BoNT/B-33/A,BoNT/C1-33/A, BoNT/D-33/A, BoNT/E-33/A, BoNT/F-33/A, BoNT/G-33/A,TeNT-33/A, as well as the modified Clostridial toxin indicated abovecomprising amino acids 10-144 of SEQ ID NO: 11; amino acids 10-144 ofSEQ ID NO: 12; amino acids 10-144 of SEQ ID NO: 13; amino acids 10-146of SEQ ID NO: 14; amino acids 10-144 of SEQ ID NO: 15; amino acids10-144 of SEQ ID NO: 16; amino acids 10-146 of SEQ ID NO: 17; aminoacids 10-141 of SEQ ID NO: 18; amino acids 10-141 of SEQ ID NO: 19;amino acids 10-141 of SEQ ID NO: 20; amino acids 151-293 of SEQ ID NO:12; amino acids 151-293 of SEQ ID NO: 13; amino acids 153-294 of SEQ IDNO: 14; amino acids 151-279 of SEQ ID NO: 15; amino acids 151-292 of SEQID NO: 16; amino acids 153-291 of SEQ ID NO: 17; amino acids 148-285 ofSEQ ID NO: 18; amino acids 148-286 of SEQ ID NO: 19; amino acids 148-286of SEQ ID NO: 20; amino acids 9-146 of SEQ ID NO: 21; amino acids 9-146of SEQ ID NO: 22; amino acids 9-146 of SEQ ID NO: 23; amino acids 9-146of SEQ ID NO: 24; amino acids 1050-1194 of SEQ ID NO: 25; amino acids1050-1199 of SEQ ID NO: 26; amino acids 1050-1194 of SEQ ID NO: 27;amino acids 1049-1198 of SEQ ID NO: 28; amino acids 1049-1197 of SEQ IDNO: 29; amino acids 1049-1197 of SEQ ID NO: 30; amino acids 1014-1163 ofSEQ ID NO: 31; amino acids 1016-1160 of SEQ ID NO: 32; amino acids1017-1166 of SEQ ID NO: 33; and amino acids 1050-1199 of SEQ ID NO: 34.

Example 3 Construction of a Multivalent Clostridial Toxin Comprising anFGF

A polynucleotide molecule encoding BoNT/A-F18 is synthesized usingstandard procedures (BlueHeron® Biotechnology, Bothell, Wash.), asdescribed in Example 1. BoNT/A-F18 is a BoNT/A modified to replace aminoacids 1111-1296 of SEQ ID NO: 1, a BoNT/A β-trefoil domain, with aminoacids 54 to 183 of SEQ ID NO: 39, a FGF-18 β-trefoil domain. If desired,an expression optimized polynucleotide molecule encoding BoNT/A-F18 canbe synthesized in order to improve expression in to a differentorganism, such as, e.g., an Escherichia coli strain, a yeast strain, aninsect cell-line or a mammalian cell line, can be done, see, e.g.,Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); andSteward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005). Thesynthesized polynucleotide molecule is verified by sequencing using BigDye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.)and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).

A polynucleotide molecule encoding BoNT/A-NtermF18 is synthesized usingstandard procedures (BlueHeron® Biotechnology, Bothell, Wash.), asdescribed in Example 1. BoNT/A-NtermF18 is a BoNT/A modified to replaceamino acids in the second binding domain (the N-terminal binding regionof the multivalent Clostridial toxin described in Example 1)corresponding to amino acids 1111-1296 of SEQ ID NO: 1, a BoNT/Aβ-trefoil domain, with amino acids 54 to 183 of SEQ ID NO: 41, a FGF-18β-trefoil domain. If desired, an expression optimized polynucleotidemolecule encoding BoNT/A-NtermF18 can be synthesized in order to improveexpression in to a different organism, such as, e.g., an Escherichiacoli strain, a yeast strain, an insect cell-line or a mammalian cellline, can be done, see, e.g., Steward, supra, PCT Patent Serial No.2005/020578 (Jun. 9, 2005); and Steward, supra, PCT Patent Serial No.2005/027917 (Aug. 3, 2005). The synthesized polynucleotide molecule isverified by sequencing using Big Dye Terminator™ Chemistry 3.1 (AppliedBiosystems, Foster City, Calif.) and an ABI 3100 sequencer (AppliedBiosystems, Foster City, Calif.).

A similar cloning strategy is used to make pUCBHB1 cloning constructsfor multivalent Clostridial toxin BoNT/B-F18, a modified BoNT/B whereamino acids 1098-1291 of SEQ ID NO: 2 are replaced with amino acids 54to 183 of SEQ ID NO: 41; multivalent Clostridial toxin BoNT/C1-F18, amodified BoNT/C1 where amino acids 1112-1291 of SEQ ID NO: 3 arereplaced with amino acids 54 to 183 of SEQ ID NO: 41; BoNT/D-F18,multivalent Clostridial toxin BoNT/D where amino acids 1099-1276 of SEQID NO: 4 are replaced with amino acids 54 to 183 of SEQ ID NO: 41;BoNT/E-F18, multivalent Clostridial toxin BoNT/E where amino acids1086-1252 of SEQ ID NO: 5 are replaced with amino acids 54 to 183 of SEQID NO: 41; BoNT/F-F18, multivalent Clostridial toxin BoNT/F where aminoacids 1106-1274 of SEQ ID NO: 6 are replaced with amino acids 54 to 183of SEQ ID NO: 41; BoNT/G-F18, multivalent Clostridial toxin BoNT/G whereamino acids 1106-1297 of SEQ ID NO: 7 are replaced with amino acids 54to 183 of SEQ ID NO: 41; multivalent Clostridial toxin TeNT-F18, TeNTwhere amino acids 1128-1315 of SEQ ID NO: 8 are replaced with aminoacids 54 to 183 of SEQ ID NO: 41; multivalent Clostridial toxinBaNT-F18, BaNT where amino acids 1095-1268 of SEQ ID NO: 9 are replacedwith amino acids 54 to 183 of SEQ ID NO: 41; multivalent Clostridialtoxin BuNT-F18, BuNT where amino acids 1086-1251 of SEQ ID NO: 10 arereplaced with amino acids 54 to 183 of SEQ ID NO: 41. Similarly, theβ-trefoil domain from a Clostridial toxin indicated above can bereplaced with a FGF β-trefoil domain comprising amino acids 26-155 ofSEQ ID NO: 35; amino acids 29-155 of SEQ ID NO: 36; amino acids 83-206of SEQ ID NO: 37; amino acids 43-172 of SEQ ID NO: 38; amino acids63-196 of SEQ ID NO: 39; and amino acids 55-183 of SEQ ID NO: 40.

To construct pET29/multivalent Clostridial toxin/BoNT/A-NtermF18, apUCBHB1/BoNT/A-F18 construct is digested with restriction endonucleasesthat 1) excise the insert comprising the open reading frame encodingBoNT/A-NtermF18; and 2) enable this insert to be operably-linked to apET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert issubcloned using a T4 DNA ligase procedure into a pET29 vector that isdigested with appropriate restriction endonucleases to yieldpET29/BoNT/A-NtermF18. The ligation mixture is transformed intochemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad,Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agarplates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C.incubator for overnight growth. Bacteria containing expressionconstructs are identified as kanamycin resistant colonies. Candidateconstructs are isolated using an alkaline lysis plasmid mini-preparationprocedure and analyzed by restriction endonuclease digest mapping todetermine the presence and orientation of the insert. This cloningstrategy yielded a pET29 expression construct comprising thepolynucleotide molecule encoding the multivalent Clostridial toxinBoNT/A-NtermF18 operably-linked to a carboxyl terminal polyhistidineaffinity binding peptide.

A similar cloning strategy is used to make pET29 expression constructscomprising a polynucleotide molecule encoding multivalent Clostridialtoxins BoNT/B-F18, BoNT/C1-F18, BoNT/D-F18, BoNT/E-F18, BoNT/F-F18,BoNT/G-F18, TeNT-F18, as well as multivalent Clostridial toxin indicatedabove comprising amino acids 26-155 of SEQ ID NO: 35; amino acids 29-155of SEQ ID NO: 36; amino acids 83-206 of SEQ ID NO: 37; amino acids43-172 of SEQ ID NO: 38; amino acids 63-196 of SEQ ID NO: 39; and aminoacids 55-183 of SEQ ID NO: 40.

Example 4 Construction of a Multivalent Clostridial NeurotoxinComprising a Binding Domain

A polynucleotide molecule encoding BoNT/A-NtermGRPP is synthesized usingstandard procedures (BlueHeron® Biotechnology, Bothell, Wash.), asdescribed in Example 1. BoNT/A-NtermGRPP is a BoNT/A modified to replaceamino acids in the second binding domain (the N-terminal binding regionof the multivalent Clostridial toxin described in Example 1)corresponding to amino acids 1111-1296 of SEQ ID NO: 1, a BoNT/Aβ-trefoil domain, with amino acids 21 to 50 of SEQ ID NO: 42, a GRPPbinding domain. If desired, an expression optimized polynucleotidemolecule encoding BoNT/A-NtermGRPP can be synthesized in order toimprove expression in to a different organism, such as, e.g., anEscherichia coli strain, a yeast strain, an insect cell-line or amammalian cell line, can be done, see, e.g., Steward, supra, PCT PatentSerial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCT PatentSerial No. 2005/027917 (Aug. 3, 2005). The synthesized polynucleotidemolecule is verified by sequencing using Big Dye Terminator™ Chemistry3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer(Applied Biosystems, Foster City, Calif.).

A similar cloning strategy is used to make pUCBHB1 cloning constructsfor multivalent Clostridial toxin BoNT/B-GRPP, a modified BoNT/B whereamino acids 1098-1291 of SEQ ID NO: 2 are replaced with amino acids 21to 50 of SEQ ID NO: 42; multivalent Clostridial toxin BoNT/C1-GRPP, amodified BoNT/C1 where amino acids 1112-1291 of SEQ ID NO: 3 arereplaced with amino acids 21 to 50 of SEQ ID NO: 42; BoNT/D-GRPP,multivalent Clostridial toxin BoNT/D where amino acids 1099-1276 of SEQID NO: 4 are replaced with amino acids 21 to 50 of SEQ ID NO: 42;BoNT/E-GRPP, multivalent Clostridial toxin BoNT/E where amino acids1086-1252 of SEQ ID NO: 5 are replaced with amino acids 21 to 50 of SEQID NO: 42; BoNT/F-GRPP, multivalent Clostridial toxin BoNT/F where aminoacids 1106-1274 of SEQ ID NO: 6 are replaced with amino acids 21 to 50of SEQ ID NO: 42; BoNT/G-GRPP, multivalent Clostridial toxin BoNT/Gwhere amino acids 1106-1297 of SEQ ID NO: 7 are replaced with aminoacids 21 to 50 of SEQ ID NO: 42; multivalent Clostridial toxinTeNT-GRPP, TeNT where amino acids 1128-1315 of SEQ ID NO: 8 are replacedwith amino acids 21 to 50 of SEQ ID NO: 42; multivalent Clostridialtoxin BaNT-GRPP, BaNT where amino acids 1095-1268 of SEQ ID NO: 9 arereplaced with amino acids 21 to 50 of SEQ ID NO: 42; multivalentClostridial toxin BuNT-GRPP, BuNT where amino acids 1086-1251 of SEQ IDNO: 10 are replaced with amino acids 21 to 50 of SEQ ID NO: 42.

Similarly, the β-trefoil domain from a Clostridial toxin indicated abovecan be replaced with a binding domain comprising, e.g., amino acids53-81 of SEQ ID NO: 42; amino acids 53-89 of SEQ ID NO: 9; amino acids98-124 of SEQ ID NO: 42; amino acids 146-178 of SEQ ID NO: 42; aminoacids 132-158 of SEQ ID NO: 43; amino acids 32-58 of SEQ ID NO: 44;amino acids 32-75 of SEQ ID NO: 44; amino acids 81-107 of SEQ ID NO: 45;amino acids 125-151 of SEQ ID NO: 45; amino acids 81-107 of SEQ ID NO:46; amino acids 124-150 of SEQ ID NO: 46; amino acids 52-78 of SEQ IDNO: 47; amino acids 52-93 of SEQ ID NO: 47; amino acids 28-54 of SEQ IDNO: 48; amino acids 76-92 of SEQ ID NO: 49; amino acids 59-92 of SEQ IDNO: 49; amino acids 41-50 of SEQ ID NO: 50; amino acids 24-50 of SEQ IDNO: 50; amino acids 99-112 of SEQ ID NO: 51; amino acids 159-193 of SEQID NO: 52; amino acids 154-194 of SEQ ID NO: 52; amino acids 35-70 ofSEQ ID NO: 53; amino acids 145-177 of SEQ ID NO: 53; amino acids 1-200of SEQ ID NO: 54; amino acids 1-150 of SEQ ID NO: 55; amino acids 1-202of SEQ ID NO: 56; amino acids 1-201 of SEQ ID NO: 57; amino acids 1-225of SEQ ID NO: 58; amino acids 123-265 of SEQ ID NO: 59; amino acids21-153 of SEQ ID NO: 60; amino acids 57-210 of SEQ ID NO: 61; aminoacids 21-99 of SEQ ID NO: 62; amino acids 31-94 of SEQ ID NO: 62; aminoacids 19-178 of SEQ ID NO: 63; amino acids 1-558 of SEQ ID NO: 64; aminoacids 1-371 of SEQ ID NO: 65; amino acids 49-118 of SEQ ID NO: 66; aminoacids 25-180 of SEQ ID NO: 67; amino acids 1-54 of SEQ ID NO: 68; aminoacids 139-257 of SEQ ID NO: 69; amino acids 129-247 of SEQ ID NO: 70;amino acids 19-257 of SEQ ID NO: 71; amino acids 81-210 of SEQ ID NO:72; amino acids 118-211 of SEQ ID NO: 73; amino acids 107-196 of SEQ IDNO: 74; amino acids 96-197 of SEQ ID NO: 74; amino acids 66-155 of SEQID NO: 75; amino acids 123-218 of SEQ ID NO: 76; amino acids 293-390 ofSEQ ID NO: 77; amino acids 317-414 of SEQ ID NO: 78; amino acids 315-412of SEQ ID NO: 79; amino acids 276-373 of SEQ ID NO: 80; amino acids296-396 of SEQ ID NO: 81; amino acids 370-472 of SEQ ID NO: 82; aminoacids 309-409 of SEQ ID NO: 83; amino acids 323-454 of SEQ ID NO: 84;amino acids 412-513 of SEQ ID NO: 85; amino acids 374-513 of SEQ ID NO:85; amino acids 330-431 of SEQ ID NO: 86; amino acids 293-431 of SEQ IDNO: 86; amino acids 301-402 of SEQ ID NO: 87; amino acids 323-424 of SEQID NO: 88; amino acids 267-372 of SEQ ID NO: 89; amino acids 327-429 ofSEQ ID NO: 90; amino acids 264-364 of SEQ ID NO: 91; amino acids 400-501of SEQ ID NO: 92; amino acids 354-455 of SEQ ID NO: 93; amino acids352-450 of SEQ ID NO: 94; amino acids 281-375 of SEQ ID NO: 95; aminoacids 376-478 of SEQ ID NO: 96; amino acids 313-407 of SEQ ID NO: 97;amino acids 211-308 of SEQ ID NO: 98; amino acids 321-426 of SEQ ID NO:99; amino acids 303-406 of SEQ ID NO: 100; amino acids 247-352 of SEQ IDNO: 101; amino acids 237-352 of SEQ ID NO: 101; amino acids 247-350 ofSEQ ID NO: 102; amino acids 262-366 of SEQ ID NO: 103; or amino acids233-366 of SEQ ID NO: 103.

To construct pET29/multivalent Clostridial toxin/BoNT/A-NtermGRPP, apUCBHB1/BoNT/A-GRPP construct is digested with restriction endonucleasesthat 1) excise the insert comprising the open reading frame encoding theBoNT/A-NtermGRPP; and 2) enable this insert to be operably-linked to apET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert issubcloned using a T4 DNA ligase procedure into a pET29 vector that isdigested with appropriate restriction endonucleases to yieldpET29/BoNT/A-NtermGRPP. The ligation mixture is transformed intochemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad,Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agarplates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C.incubator for overnight growth. Bacteria containing expressionconstructs are identified as kanamycin resistant colonies. Candidateconstructs are isolated using an alkaline lysis plasmid mini-preparationprocedure and analyzed by restriction endonuclease digest mapping todetermine the presence and orientation of the insert. This cloningstrategy yielded a pET29 expression construct comprising thepolynucleotide molecule encoding the multivalent Clostridial toxinBoNT/A-NtermGRPP operably-linked to a carboxyl terminal polyhistidineaffinity binding peptide.

A similar cloning strategy is used to make pET29 expression constructscomprising a polynucleotide molecule encoding multivalent Clostridialtoxins BoNT/B-GRPP, BoNT/C1-GRPP, BoNT/D-GRPP, BoNT/E-GRPP, BoNT/F-GRPP,BoNT/G-GRPP, TeNT-GRPP, BaNT-GRPP, BuNT-GRPP, as well as multivalentClostridial toxin indicated above comprising, e.g., amino acids 53-81 ofSEQ ID NO: 42; amino acids 53-89 of SEQ ID NO: 9; amino acids 98-124 ofSEQ ID NO: 42; amino acids 146-178 of SEQ ID NO: 42; amino acids 132-158of SEQ ID NO: 43; amino acids 32-58 of SEQ ID NO: 44; amino acids 32-75of SEQ ID NO: 44; amino acids 81-107 of SEQ ID NO: 45; amino acids125-151 of SEQ ID NO: 45; amino acids 81-107 of SEQ ID NO: 46; aminoacids 124-150 of SEQ ID NO: 46; amino acids 52-78 of SEQ ID NO: 47;amino acids 52-93 of SEQ ID NO: 47; amino acids 28-54 of SEQ ID NO: 48;amino acids 76-92 of SEQ ID NO: 49; amino acids 59-92 of SEQ ID NO: 49;amino acids 41-50 of SEQ ID NO: 50; amino acids 24-50 of SEQ ID NO: 50;amino acids 99-112 of SEQ ID NO: 51; amino acids 159-193 of SEQ ID NO:52; amino acids 154-194 of SEQ ID NO: 52; amino acids 35-70 of SEQ IDNO: 53; amino acids 145-177 of SEQ ID NO: 53; amino acids 1-200 of SEQID NO: 54; amino acids 1-150 of SEQ ID NO: 55; amino acids 1-202 of SEQID NO: 56; amino acids 1-201 of SEQ ID NO: 57; amino acids 1-225 of SEQID NO: 58; amino acids 123-265 of SEQ ID NO: 59; amino acids 21-153 ofSEQ ID NO: 60; amino acids 57-210 of SEQ ID NO: 61; amino acids 21-99 ofSEQ ID NO: 62; amino acids 31-94 of SEQ ID NO: 62; amino acids 19-178 ofSEQ ID NO: 63; amino acids 1-558 of SEQ ID NO: 64; amino acids 1-371 ofSEQ ID NO: 65; amino acids 49-118 of SEQ ID NO: 66; amino acids 25-180of SEQ ID NO: 67; amino acids 1-54 of SEQ ID NO: 68; amino acids 139-257of SEQ ID NO: 69; amino acids 129-247 of SEQ ID NO: 70; amino acids19-257 of SEQ ID NO: 71; amino acids 81-210 of SEQ ID NO: 72; aminoacids 118-211 of SEQ ID NO: 73; amino acids 107-196 of SEQ ID NO: 74;amino acids 96-197 of SEQ ID NO: 74; amino acids 66-155 of SEQ ID NO:75; amino acids 123-218 of SEQ ID NO: 76; amino acids 293-390 of SEQ IDNO: 77; amino acids 317-414 of SEQ ID NO: 78; amino acids 315-412 of SEQID NO: 79; amino acids 276-373 of SEQ ID NO: 80; amino acids 296-396 ofSEQ ID NO: 81; amino acids 370-472 of SEQ ID NO: 82; amino acids 309-409of SEQ ID NO: 83; amino acids 323-454 of SEQ ID NO: 84; amino acids412-513 of SEQ ID NO: 85; amino acids 374-513 of SEQ ID NO: 85; aminoacids 330-431 of SEQ ID NO: 86; amino acids 293-431 of SEQ ID NO: 86;amino acids 301-402 of SEQ ID NO: 87; amino acids 323-424 of SEQ ID NO:88; amino acids 267-372 of SEQ ID NO: 89; amino acids 327-429 of SEQ IDNO: 90; amino acids 264-364 of SEQ ID NO: 91; amino acids 400-501 of SEQID NO: 92; amino acids 354-455 of SEQ ID NO: 93; amino acids 352-450 ofSEQ ID NO: 94; amino acids 281-375 of SEQ ID NO: 95; amino acids 376-478of SEQ ID NO: 96; amino acids 313-407 of SEQ ID NO: 97; amino acids211-308 of SEQ ID NO: 98; amino acids 321-426 of SEQ ID NO: 99; aminoacids 303-406 of SEQ ID NO: 100; amino acids 247-352 of SEQ ID NO: 101;amino acids 237-352 of SEQ ID NO: 101; amino acids 247-350 of SEQ ID NO:102; amino acids 262-366 of SEQ ID NO: 103; or amino acids 233-366 ofSEQ ID NO: 103.

Example 5 Construction of a Multivalent Clostridial NeurotoxinComprising a Binding Domain

A polynucleotide molecule encoding BoNT/A-Noci is synthesized usingstandard procedures (BlueHeron® Biotechnology, Bothell, Wash.), asdescribed in Example 1. BoNT/A-Noci is a BoNT/A modified to replaceamino acids 874-1296 of SEQ ID NO: 1, a BoNT/A β-trefoil domain, with anociceptin-RK targeting domain. If desired, an expression optimizedpolynucleotide molecule encoding BoNT/A-Noci can be synthesized in orderto improve expression in to a different organism, such as, e.g., anEscherichia coli strain, a yeast strain, an insect cell-line or amammalian cell line, can be done, see, e.g., Steward, supra, PCT PatentSerial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCT PatentSerial No. 2005/027917 (Aug. 3, 2005). The synthesized polynucleotidemolecule is verified by sequencing using Big Dye Terminator™ Chemistry3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer(Applied Biosystems, Foster City, Calif.).

A polynucleotide molecule encoding BoNT/A-NtermNoci is synthesized usingstandard procedures (BlueHeron® Biotechnology, Bothell, Wash.), asdescribed in Example 1. BoNT/A-NtermNoci is a BoNT/A modified to replaceamino acids in the second binding domain (the N-terminal binding regionof the multivalent Clostridial toxin described in Example 1)corresponding to amino acids 874-1296 of SEQ ID NO: 1, a BoNT/Aβ-trefoil domain, with SEQ ID NO: 152, a nociceptin-RK binding domain.If desired, an expression optimized polynucleotide molecule encodingBoNT/A-NtermNoci can be synthesized in order to improve expression in toa different organism, such as, e.g., an Escherichia coli strain, a yeaststrain, an insect cell-line or a mammalian cell line, can be done, see,e.g., Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005);and Steward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005).The synthesized polynucleotide molecule is verified by sequencing usingBig Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City,Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City,Calif.).

A similar cloning strategy is used to make pUCBHB1 cloning constructsfor multivalent Clostridial toxin BoNT/B-Noci, a modified BoNT/B whereamino acids 861-1291 of SEQ ID NO: 2 are replaced with SEQ ID NO: 152;multivalent Clostridial toxin BoNT/C1-Noci, a modified BoNT/C1 whereamino acids 869-1291 of SEQ ID NO: 3 are replaced with SEQ ID NO: 152;BoNT/D-Noci, multivalent Clostridial toxin BoNT/D where amino acids865-1276 of SEQ ID NO: 4 are replaced with SEQ ID NO: 152; BoNT/E-Noci,multivalent Clostridial toxin BoNT/E where amino acids 848-1252 of SEQID NO: 5 are replaced with SEQ ID NO: 152; BoNT/F-Noci, multivalentClostridial toxin BoNT/F where amino acids 867-1274 of SEQ ID NO: 6 arereplaced with SEQ ID NO: 152; BoNT/G-Noci, multivalent Clostridial toxinBoNT/G where amino acids 866-1297 of SEQ ID NO: 7 are replaced with SEQID NO: 152; multivalent Clostridial toxin TeNT-Noci, TeNT where aminoacids 882-1315 of SEQ ID NO: 8 are replaced with SEQ ID NO: 152;multivalent Clostridial toxin BaNT-Noci, BaNT where amino acids 858-1268of SEQ ID NO: 9 are replaced with SEQ ID NO: 152; multivalentClostridial toxin BuNT-Noci, BuNT where amino acids 848-1251 of SEQ IDNO: 10 are replaced with SEQ ID NO: 152.

Similarly, the β-trefoil domain from a Clostridial toxin indicated abovecan be replaced with a binding domain comprising, e.g., SEQ ID NO: 104;SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 114, SEQ IDNO: 115, SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118,SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ IDNO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127,SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ IDNO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136,SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ IDNO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145,SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ IDNO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154,SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ IDNO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163,SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ IDNO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172,SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ IDNO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181,SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ IDNO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190,SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ IDNO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199,SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ IDNO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208,SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ IDNO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217,SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ IDNO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226,SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ IDNO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239,SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ IDNO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248,SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, or SEQID NO: 253.

Similarly, the β-trefoil domain from a Clostridial toxin indicated abovecan be replaced with a binding domain comprising, e.g., amino acids1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ IDNO: 108; amino acids 1-12, amino acids 6-22, amino acids 8-22 or aminoacids 1-22 of SEQ ID NO: 109; amino acids 1-12, amino acids 6-22, aminoacids 8-22 or amino acids 1-22 of SEQ ID NO: 110; amino acids 1-12,amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO:111; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids1-22 of SEQ ID NO: 112; amino acids 1-12, amino acids 6-22, amino acids8-22 or amino acids 1-22 of SEQ ID NO: 113; amino acids 20-58 of SEQ IDNO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204,SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ IDNO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214or SEQ ID NO: 215; amino acids 26-58 of SEQ ID NO: 200, SEQ ID NO: 201,SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ IDNO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211,SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214 or SEQ ID NO: 215; aminoacids 47-58 of SEQ ID NO: 200, SEQ ID NO: 210 or SEQ ID NO: 214; oramino acids 51-58 of SEQ ID NO: 200; amino acids 42-47, amino acids42-55, amino acids 29-64 or amino acids 1-64 of SEQ ID NO: 231; aminoacids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 ofSEQ ID NO: 232; amino acids 39-44, amino acids 39-52, amino acids 26-60or amino acids 1-60 of SEQ ID NO: 233; amino acids 48-53, amino acids48-61, amino acids 35-70 or amino acids 1-70 of SEQ ID NO: 234.

To construct pET29/multivalent Clostridial toxin/BoNT/A-NtermGRPP, apUCBHB1/BoNT/A-Noci construct is digested with restriction endonucleasesthat 1) excise the insert comprising the open reading frame encoding theBoNT/A-NtermNoci; and 2) enable this insert to be operably-linked to apET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert issubcloned using a T4 DNA ligase procedure into a pET29 vector that isdigested with appropriate restriction endonucleases to yieldpET29/BoNT/A-NtermNoci. The ligation mixture is transformed intochemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad,Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agarplates (pH 7.0) containing 50 μg/mL of kanamycin, and placed in a 37° C.incubator for overnight growth. Bacteria containing expressionconstructs are identified as kanamycin resistant colonies. Candidateconstructs are isolated using an alkaline lysis plasmid mini-preparationprocedure and analyzed by restriction endonuclease digest mapping todetermine the presence and orientation of the insert. This cloningstrategy yielded a pET29 expression construct comprising thepolynucleotide molecule encoding the multivalent Clostridial toxinBoNT/A-NtermNoci operably-linked to a carboxyl terminal polyhistidineaffinity binding peptide.

A similar cloning strategy is used to make pET29 expression constructscomprising a polynucleotide molecule encoding multivalent Clostridialtoxins BoNT/B-Noci, BoNT/C1-Noci, BoNT/D-Noci, BoNT/E-Noci, BoNT/F-Noci,BoNT/G-Noci, TeNT-Noci, BaNT-Noci, BuNT-Noci, as well as multivalentClostridial toxin indicated above comprising, e.g., SEQ ID NO: 104; SEQID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 114, SEQ ID NO:115, SEQ ID NO: 291, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO:141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO:150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO:159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO:168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO:177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO:186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO:195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO:204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO:213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO:222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO:235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO:244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO:253; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids1-22 of SEQ ID NO: 108; amino acids 1-12, amino acids 6-22, amino acids8-22 or amino acids 1-22 of SEQ ID NO: 109; amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 110;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 111; amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 112; amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 113; aminoacids 20-58 of SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ IDNO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 208,SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ IDNO: 213, SEQ ID NO: 214 or SEQ ID NO: 215; amino acids 26-58 of SEQ IDNO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204,SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ IDNO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214or SEQ ID NO: 215; amino acids 47-58 of SEQ ID NO: 200, SEQ ID NO: 210or SEQ ID NO: 214; or amino acids 51-58 of SEQ ID NO: 200; amino acids42-47, amino acids 42-55, amino acids 29-64 or amino acids 1-64 of SEQID NO: 231; amino acids 35-40, amino acids 35-48, amino acids 24-59 oramino acids 1-59 of SEQ ID NO: 232; amino acids 39-44, amino acids39-52, amino acids 26-60 or amino acids 1-60 of SEQ ID NO: 233; aminoacids 48-53, amino acids 48-61, amino acids 35-70 or amino acids 1-70 ofSEQ ID NO: 234

Example 6 Construction of a Multivalent Clostridial NeurotoxinComprising Three CCK-A Binding Sites

The following example illustrates how to make a retargeted multivalentClostridial toxin comprising three CCK-A binding sites.

A polynucleotide molecule is constructed as described in Example 1above, encoding BoNT/A with the native binding site replaced with 10repeats of the CCK 58 amino acid sequence (see U.S. Pat. No. 6,843,998,hereby incorporated by reference herein in its entirety) and with thesame CCK 58 sequence repeated twice at the N-terminus of the singlechain. The two N-terminal repeats are constructed to be separated by ashort amino acid region comprising the loop region of BoNT/A, and twocysteine residues spanning the protease sensitive site. The multivalentClostridial toxin is termed multivalent Clostridial toxinBoNT/A/C(CCK)N(CCKx2). The basic architecture of the amino acidconstruct is given in FIG. 2A.

If desired, an expression-optimized polynucleotide molecule encodingmultivalent Clostridial toxin BoNT/A/C(CCK)N(CCKx2) can be synthesizedin order to improve expression in to a different organism, such as,e.g., an Escherichia coli strain, a yeast strain, an insect cell-line ora mammalian cell line, can be done, see, e.g., Steward, supra, PCTPatent Serial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCTPatent Serial No. 2005/027917 (Aug. 3, 2005). The synthesizedpolynucleotide molecule is verified by sequencing using Big DyeTerminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) andan ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).

A similar cloning strategy is used to make pUCBHB1 cloning constructsfor C(CCK)N(CCKx2) multivalent Clostridial toxin's in which thetranslocation and endopeptidase domains are independently selected fromBoNT/B, C1, D, E, F, G, or TeNT.

To construct pET29/multivalent Clostridial toxin BoNT/A/C(CCK)N(CCKx2),a pUCBHB1/multivalent Clostridial toxin BoNT/A/C(CCK)N(CCKx2) constructis digested with restriction endonucleases that 1) excise the insertcomprising the open reading frame encoding multivalent Clostridial toxinBoNT/A/C(CCK)N(CCKx2); and 2) enable this insert to be operably-linkedto a pET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insertis subcloned using a T4 DNA ligase procedure into a pET29 vector that isdigested with appropriate restriction endonucleases to yieldpET29/multivalent Clostridial toxin BoNT/A/C(CCK)N(CCKx2). The ligationmixture is transformed into chemically competent E. coli DH5α cells(Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL ofKanamycin, and placed in a 37° C. incubator for overnight growth.Bacteria containing expression constructs are identified as Kanamycinresistant colonies. Candidate constructs are isolated using an alkalinelysis plasmid mini-preparation procedure and analyzed by restrictionendonuclease digest mapping to determine the presence and orientation ofthe insert. This cloning strategy yielded a pET29 expression constructcomprising the polynucleotide molecule encoding multivalent Clostridialtoxin BoNT/A/C(CCK)N(CCKx2), operably-linked to a carboxyl terminalpolyhistidine affinity binding peptide.

A similar cloning strategy is used to make pET29 expression constructscomprising a polynucleotide molecule encoding any combination oftranslocation and endopeptidase domains derived from BoNT/A, B, C1, D,E, F, G or TeNT with CCK 58 domains similar positioned as in multivalentClostridial toxin BoNT/A/C(CCK)N(CCKx2).

Example 7 Purification and Quantification of Modified Clostridial Toxins

The following example illustrates methods useful for purification andquantification of any modified Clostridial toxins disclosed in thepresent specification.

For immobilized metal affinity chromatography (IMAC) proteinpurification, E. coli BL21 (DE3) cell pellets used to express a modifiedClostridial toxin, as described in Example 7, are resuspended in ColumnBinding Buffer (25 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonicacid) (HEPES), pH 7.8; 500 mM sodium chloride; 10 mM imidazole; 2xProtease Inhibitor Cocktail Set III (EMD Biosciences-Calbiochem, SanDiego Calif.); 5 units/mL of Benzonase (EMD Biosciences-Novagen,Madison, Wis.); 0.1% (v/v) Triton-X® 100, 4-octylphenol polyethoxylate;10% (v/v) glycerol), and then are transferred to a cold Oakridgecentrifuge tube. The cell suspension is sonicated on ice (10-12 pulsesof 10 seconds at 40% amplitude with 60 seconds cooling intervals on aBranson Digital Sonifier) in order to lyse the cells and then iscentrifuged (16,000 rpm at 4° C. for 20 minutes) to clarify the lysate.An immobilized metal affinity chromatography column is prepared using a20 mL Econo-Pac column support (Bio-Rad Laboratories, Hercules, Calif.)packed with 2.5-5.0 mL of TALON™ SuperFlow Co²⁺ affinity resin (BDBiosciences-Clontech, Palo Alto, Calif.), which is then equilibrated byrinsing with 5 column volumes of deionized, distilled water, followed by5 column volumes of Column Binding Buffer. The clarified lysate isapplied slowly to the equilibrated column by gravity flow (approximately0.25-0.3 mL/minute). The column is then washed with 5 column volumes ofColumn Wash Buffer (N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonicacid) (HEPES), pH 7.8; 500 mM sodium chloride; 10 mM imidazole; 0.1%(v/v) Triton-X® 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol).The modified Clostridial toxin is eluted with 20-30 mL of Column ElutionBuffer (25 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)(HEPES), pH 7.8; 500 mM sodium chloride; 500 mM imidazole; 0.1% (v/v)Triton-X® 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol) and iscollected in approximately twelve 1 mL fractions. The amount of modifiedClostridial toxin contained in each elution fraction is determined by aBradford dye assay. In this procedure, 20 μL aliquots of each 1.0 mLfraction is combined with 200 μL of Bio-Rad Protein Reagent (Bio-RadLaboratories, Hercules, Calif.), diluted 1 to 4 with deionized,distilled water, and then the intensity of the colorimetric signal ismeasured using a spectrophotometer. The five fractions with thestrongest signal are considered the elution peak and are combinedtogether. Total protein yield is determined by estimating the totalprotein concentration of the pooled peak elution fractions using bovinegamma globulin as a standard (Bio-Rad Laboratories, Hercules, Calif.).

For purification of a modified Clostridial toxin using a FPLC desaltingcolumn, a HiPrep™ 26/10 size exclusion column (Amersham Biosciences,Piscataway, N.J.) is pre-equilibrated with 80 mL of 4° C. Column Buffer(50 mM sodium phosphate, pH 6.5). After the column is equilibrated, amodified Clostridial toxin sample is applied to the size exclusioncolumn with an isocratic mobile phase of 4° C. Column Buffer and at aflow rate of 10 mL/minute using a BioLogic DuoFlow chromatography system(Bio-Rad Laboratories, Hercules, Calif.). The desalted modifiedClostridial toxin sample is collected as a single fraction ofapproximately 7-12 mL.

For purification of a modified Clostridial toxin using a FPLC ionexchange column, a modified Clostridial toxin sample that has beendesalted following elution from an IMAC column is applied to a 1 mL Q1™anion exchange column (Bio-Rad Laboratories, Hercules, Calif.) using aBioLogic DuoFlow chromatography system (Bio-Rad Laboratories, Hercules,Calif.). The sample is applied to the column in 4° C. Column Buffer (50mM sodium phosphate, pH 6.5) and is eluted by linear gradient with 4° C.Elution Buffer (50 mM sodium phosphate, 1 M sodium chloride, pH 6.5) asfollows: step 1, 5.0 mL of 5% Elution Buffer at a flow rate of 1mL/minute; step 2, 20.0 mL of 5-30% Elution Buffer at a flow rate of 1mL/minute; step 3, 2.0 mL of 50% Elution Buffer at a flow rate of 1.0mL/minute; step 4, 4.0 mL of 100% Elution Buffer at a flow rate of 1.0mL/minute; and step 5, 5.0 mL of 0% Elution Buffer at a flow rate of 1.0mL/minute. Elution of modified Clostridial toxin from the column ismonitored at 280, 260, and 214 nm, and peaks absorbing above a minimumthreshold (0.01 au) at 280 nm are collected. Most of the modifiedClostridial toxin will elute at a sodium chloride concentration ofapproximately 100 to 200 mM. Average total yields of modifiedClostridial toxin will be determined by a Bradford assay.

Expression of a modified Clostridial toxin is analyzed by polyacrylamidegel electrophoresis. Samples purified using the procedure describedabove are added to 2×LDS Sample Buffer (Invitrogen, Inc, Carlsbad,Calif.) and are separated by MOPS polyacrylamide gel electrophoresisusing NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels(Invitrogen, Inc, Carlsbad, Calif.) under denaturing, reducingconditions. Gels are stained with SYPRO® Ruby (Bio-Rad Laboratories,Hercules, Calif.) and the separated polypeptides are imaged using aFluor-S MAX Multilmager (Bio-Rad Laboratories, Hercules, Calif.) forquantification of modified Clostridial toxin expression levels. The sizeand amount of modified Clostridial toxin is determined by comparison toMagicMark™ protein molecular weight standards (Invitrogen, Inc,Carlsbad, Calif.).

Expression of modified Clostridial toxin is also analyzed by Westernblot analysis. Protein samples purified using the procedure describedabove are added to 2×LDS Sample Buffer (Invitrogen, Inc, Carlsbad,Calif.) and are separated by MOPS polyacrylamide gel electrophoresisusing NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels(Invitrogen, Inc, Carlsbad, Calif.) under denaturing, reducingconditions. Separated polypeptides are transferred from the gel ontopolyvinylidene fluoride (PVDF) membranes (Invitrogen, Inc, Carlsbad,Calif.) by Western blotting using a Trans-Blot® SD semi-dryelectrophoretic transfer cell apparatus (Bio-Rad Laboratories, Hercules,Calif.). PVDF membranes are blocked by incubating at room temperaturefor 2 hours in a solution containing 25 mM Tris-Buffered Saline (25 mM2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid (Tris-HCl) (pH7.4), 137 mM sodium chloride, 2.7 mM potassium chloride), 0.1%TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate, 2% bovine serumalbumin, 5% nonfat dry milk. Blocked membranes are incubated at 4° C.for overnight in Tris-Buffered Saline TWEEN-20® (25 mM Tris-BufferedSaline, 0.1% TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate)containing appropriate primary antibodies as a probe. Primary antibodyprobed blots are washed three times for 15 minutes each time inTris-Buffered Saline TWEEN-20. Washed membranes are incubated at roomtemperature for 2 hours in Tris-Buffered Saline TWEEN-20® containing anappropriate immunoglobulin G antibody conjugated to horseradishperoxidase as a secondary antibody. Secondary antibody-probed blots arewashed three times for 15 minutes each time in Tris-Buffered SalineTWEEN-20®. Signal detection of the labeled modified Clostridial toxinare visualized using the ECL Plus™ Western Blot Detection System(Amersham Biosciences, Piscataway, N.J.) and are imaged with a Typhoon9410 Variable Mode Imager (Amersham Biosciences, Piscataway, N.J.) forquantification of modified Clostridial toxin expression levels.

Example 8 Treatment of Hyperhidrosis Using a Multivalent ClostridialToxin

A 32-year-old woman presents complaining with chronic and excessiveperspiring under the arms and in the palm of the hands. Clinicalexamination reveals a high degree of sweating under the arms, and stainson clothing in the same area.

The patient is injected in the eccrine glands under one arm with anapproximately minimum effective dose (15 drops) of BOTOX®. The samepatient is injected in the eccrine glands under the other arm with 7drops of the Multivalent Clostridial Neurotoxin Derivative of Example 1.

The patient is observed one week later. Examination reveals thatexcessive sweating under the arms has been deceased by 85-95% in bothcases, despite the fact that the multivalent Clostridial toxin wasadministered at less than 50% of the BOTOX® dosage.

Example 9 Treatment of Acute Pancreatitis with a Multivalent ClostridialToxin

A 55 year-old man with a history of alcoholism presents with nausea,loss of appetite and severe abdominal pain radiating to the back.Examination reveals that the patient suffers from acute pancreatitiswith a Balthazar Score of Grade D (with fluid collection in a singlepancreatic location). The acute and advancing pancreatic necrosisthreatens the patient's life.

The patient is administered the multivalent Clostridial toxin of Example3 in an effective dose by injection directly into the pancreatic acini.Within 48 hours, there is a halt in the progression of the patient'sdeterioration. Within two weeks the acute pain has been relieved and thepatient is able to take oral nourishment.

Although aspects of the present invention have been described withreference to the disclosed embodiments, one skilled in the art willreadily appreciate that the specific examples disclosed are onlyillustrative of these aspects and in no way limit the present invention.Various modifications can be made without departing from the spirit ofthe present invention.

Any and all patents, publications, patent applications, and nucleotideand/or amino acid sequences referred to by accession numbers cited inthis specification are hereby incorporated by reference as part of thisspecification.

Each and every feature described herein, and each and every combinationof two or more of such features, is included within the scope of thepresent invention provided that the features included in such acombination are not mutually inconsistent.

These and other aspects of the present invention are set forth in thefollowing claims.

1. A multivalent Clostridial toxin comprising: a) a Clostridial toxinenzymatic domain capable of executing an enzymatic target modificationstep of a Clostridial toxin intoxication process; b) a Clostridial toxintranslocation domain capable of executing a translocation step of aClostridial toxin intoxication process; c) a first binding domaincapable of executing a cell binding step of a Clostridial toxinintoxication process by selectively binding a first cell surfacereceptor displayed by the target cell; and d) a second binding domaincapable of executing a cell binding step of a Clostridial toxinintoxication process by selectively binding a second cell surfacereceptor displayed by the target cell; and e) a protease cleavage site,wherein cleavage of the protease cleavage site converts the single-chainform of the modified Clostridial toxin into the di-chain form.
 2. Themultivalent Clostridial toxin according to claim 1, wherein themultivalent Clostridial toxin comprises amino to carboxyl linearorganization comprising a) a binding domain 1, a translocation domain, abinding domain 2, a protease cleavage site and an enzymatic domain, b)an enzymatic domain, a protease cleavage site, a binding domain 1, atranslocation domain and a binding domain 2, c) an enzymatic domain, aprotease cleavage site, a translocation domain, a binding domain 1 and abinding domain 2, d) an enzymatic domain, a protease cleavage site, atranslocation domain, a binding domain 2 and a binding domain 1, e) abinding domain 1, an enzymatic domain, a protease cleavage site, atranslocation domain and a binding domain 2, f) a translocation domain,a binding domain 2, a protease cleavage site, a binding domain 1 and anenzymatic domain, g) a translocation domain, a binding domain 2, aprotease cleavage site, an enzymatic domain and a binding domain 1, orh) a binding domain 2, a translocation domain, a protease cleavage site,a binding domain 1 and an enzymatic domain.
 3. The multivalentClostridial toxin according to claim 1, wherein the protease cleavagesite is an endogenous protease cleavage site or an exogenous proteasecleavage site.
 4. The multivalent Clostridial toxin according to claim3, wherein the endogenous protease cleavage site is selected from thegroup consisting of a BoNT/A di-chain loop protease cleavage site, aBoNT/B di-chain loop protease cleavage site, a BoNT/C1 di-chain loopprotease cleavage site, a BoNT/D di-chain loop protease cleavage site, aBoNT/E di-chain loop protease cleavage site, a BoNT/F di-chain loopprotease cleavage site, a BoNT/G di-chain loop protease cleavage siteand a TeNT di-chain loop protease cleavage site.
 5. The multivalentClostridial toxin according to claim 3, wherein the exogenous proteasecleavage site is selected from the group consisting of a bovineenterokinase protease cleavage site, a Tobacco Etch Virus proteasecleavage site, a Human Rhinovirus 3C protease cleavage site, aSUMO/ULP-1 protease cleavage site, a Thrombin protease cleavage site,and a Factor Xa protease cleavage site.
 6. The multivalent Clostridialtoxin according to claim 1, wherein the Clostridial toxin enzymaticdomain is selected from the group consisting of a BoNT/A enzymaticdomain, a BoNT/B enzymatic domain, a BoNT/C1 enzymatic domain, a BoNT/Denzymatic domain, a BoNT/E enzymatic domain, a BoNT/F enzymatic domain,a BoNT/G enzymatic domain, a TeNT enzymatic domain, a BaNT enzymaticdomain and a BuNT enzymatic domain.
 7. The multivalent Clostridial toxinaccording claim 1, wherein the Clostridial toxin translocation domain isselected from the group consisting of a BoNT/A translocation domain, aBoNT/B translocation domain, a BoNT/C1 translocation domain, a BoNT/Dtranslocation domain, a BoNT/E translocation domain, a BoNT/Ftranslocation domain, a BoNT/G translocation domain, a TeNTtranslocation domain, a BaNT translocation domain and a BuNTtranslocation domain.
 8. The multivalent Clostridial toxin accordingclaim 1, wherein the first binding domain comprises a binding domainselected from the group consisting of a Clostridial toxin bindingdomain, a Clostridial non-toxin associated protein β-trefoil domain andan FGF β-trefoil domain.
 9. The multivalent Clostridial toxin accordingclaim 1, wherein the first binding domain comprises a binding domainselected from the group consisting of a glucagon like hormone, aneurohormone, a neuroregulatory cytokine, a neurotrophin, a growthfactor, and an axon guidance signaling molecule.
 10. The multivalentClostridial toxin according claim 1, wherein the first binding domaincomprises a binding domain selected from the group consisting of anopioid peptide, a melanocortin peptide, a galanin peptide, a graninpeptide, a tachykinin peptide, a cholecystokinin peptide, a NeuropeptideY related peptide, a kinin peptide, a protease activated receptor (PAR)peptide, a somatostatin peptide, a leukemia inhibitor factor peptide,and an interleukin-1 peptide.
 11. The multivalent Clostridial toxinaccording claim 1, wherein the first binding domain comprises a PTD. 12.The multivalent Clostridial toxin according to claim 11, wherein the PTDis selected from the group consisting of a herpes simplex virus type 1VP22 protein translocating sequence, a SV-40 virus large T translocatingsequence, a TAT translocating sequence, an adenovirus translocatingsequence, a synthetic integrin binding domain translocating sequence, aKaposi fibroblast growth factor membrane translocating sequence, anuclear localization signal, a Transportan translocating sequence, aciliary neurotrophic factor translocating sequence, a caveolin, aninterleukin 1-β translocating sequence, a thioredoxin translocatingsequence, a fibroblast growth factor-1 translocating sequence, afibroblast growth factor-2 translocating sequence, an integrin β1translocating sequence, an integrin β3 translocating sequence, alactoferrin translocating sequence, a homeodomain translocatingsequence, like, a penetratin translocating sequence, an Engrailed-1translocating sequence, an Engrailed-2 translocating sequence, a Hoxa-5translocating sequence, a Hoxb-4 translocating sequence, and a Hoxc-8translocating sequence.
 13. A polynucleotide molecule encoding amultivalent Clostridial toxin according to claim
 1. 14. Thepolynucleotide molecule according to claim 13, wherein thepolynucleotide molecule is an expression construct.
 15. A method ofproducing a multivalent Clostridial toxin comprising the step ofexpressing in a cell a polynucleotide according to claim 14
 16. A methodof producing a multivalent Clostridial toxin comprising the steps of a)introducing in a cell an expression construct comprising apolynucleotide according to claim 14; and b) expressing the expressionconstruct in the cell.